Spiro-oxindole mdm2 antagonists

ABSTRACT

Provided herein are compounds, compositions, and methods in the field of medicinal chemistry. The compounds and compositions provided herein relate to spiro-oxindoles which function as antagonists of the interaction between p53 and MDM2, and their use as therapeutics for the treatment of cancer and other diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of pending U.S. patent applicationSer. No. 13/944,587, filed Jul. 17, 2013, which is a continuation ofpending U.S. patent application Ser. No. 12/945,511, filed Nov. 12,2010, now allowed as U.S. Pat. No. 8,518,984, which claims priority toexpired U.S. Provisional Patent Application No. 61/260,685, filed Nov.12, 2009, and expired U.S. Provisional Patent Application No.61/263,662, filed Nov. 23, 2009.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under CA121279;CA069568; and CA097248 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND

The aggressive cancer cell phenotype is the result of a variety ofgenetic and epigenetic alterations leading to deregulation ofintracellular signaling pathways (Ponder, Nature 411:336 (2001)). Cancercells typically fail to execute an apoptotic program, and lack ofappropriate apoptosis due to defects in the normal apoptosis machineryis considered a hallmark of cancer (Lowe et al., Carcinogenesis 21:485(2000)). The inability of cancer cells to execute an apoptotic programdue to defects in the normal apoptotic machinery is often associatedwith an increase in resistance to chemotherapy, radiation, orimmunotherapy-induced apoptosis. Primary or acquired resistance of humancancer of different origins to current treatment protocols due toapoptosis defects is a major problem in current cancer therapy (Lowe etal., Carcinogenesis 21:485 (2000); Nicholson, Nature 407:810 (2000)).Accordingly, current and future efforts towards designing and developingnew molecular target-specific anticancer therapies to improve survivaland quality of life of cancer patients must include strategies thatspecifically target cancer cell resistance to apoptosis.

The p53 tumor suppressor plays a central role in controlling cell cycleprogression, senescence, and apoptosis (Vogelstein et al., Nature408:307 (2000); Goberdhan, Cancer Cell 7:505 (2005)). MDM2 and p53 arepart of an auto-regulatory feed-back loop (Wu et al., Genes Dev. 7:1126(1993)). MDM2 is transcriptionally activated by p53 and MDM2, in turn,inhibits p53 activity by at least three mechanisms (Wu et al., GenesDev. 7:1126 (1993). First, MDM2 protein directly binds to the p53transactivation domain and thereby inhibits p53-mediatedtransactivation. Second, MDM2 protein contains a nuclear export signalsequence, and upon binding to p53, induces the nuclear export of p53,preventing p53 from binding to the targeted DNAs. Third, MDM2 protein isan E3 ubiquitin ligase and upon binding to p53 is able to promote p53degradation.

Although high-affinity peptide-based inhibitors of MDM2 have beensuccessfully designed in the past (Garcia-Echeverria et al., Med. Chem.43:3205 (2000)), these inhibitors are not suitable therapeutic moleculesbecause of their poor cell permeability and in vivo bioavailability.Despite intensive efforts by the pharmaceutical industry, highthroughput screening strategies have had very limited success inidentifying potent, non-peptide small molecule inhibitors. Accordingly,there is a need for non-peptide, drug-like, small molecule inhibitors ofthe p53-MDM2 interaction.

The structural basis of the interaction p53 and MDM2 has beenestablished by x-ray crystallography (Kussie et al., Science 274:948(1996)).

Spiro-oxindole-based antagonists of the p53-MDM2 interaction aredescribed in U.S. Pat. Nos. 7,759,383 B2 and 7,737,174 B2.

SUMMARY OF THE INVENTION

The present disclosure contemplates that exposure of animals sufferingfrom cancer to therapeutically effective amounts of drug(s) (e.g., smallmolecules) that increase the function(s) of p53 and p53-related proteins(e.g., p63, p73) inhibits the growth of cancer cells or supportingcells. In some embodiments, the compounds provided herein inhibit theinteraction between p53 or p53-related proteins and MDM2 or MDM2-relatedproteins (e.g., MDMX). Inhibiting the interaction between p53 orp53-related proteins and MDM2 or MDM2-related proteins inhibits thegrowth of cancer cells or supporting cells and/or renders such cells asa population more susceptible to the cell death-inducing activity ofcancer therapeutic drugs or radiation therapies. In some embodiments,the inhibitors provided herein prolong the half-life of p53 byinterfering with the p53-MDM2 interaction that would normally promotedegradation of p53 The compounds provided herein satisfy an unmet needfor the treatment of multiple cancer types, either when administered asmonotherapy to induce senescence, cell growth inhibition, apoptosisand/or cell cycle arrest in cancer cells, or when administered in atemporal relationship with additional agent(s), such as other celldeath-inducing or cell cycle disrupting cancer therapeutic drugs orradiation therapies (combination therapies), so as to render a greaterproportion of the cancer cells or supportive cells susceptible toexecuting the apoptosis program compared to the corresponding proportionof cells in an animal treated only with the cancer therapeutic drug orradiation therapy alone.

In some embodiments, treatment of animals with a therapeuticallyeffective amount of one or more compounds provided herein and ananticancer agent produces a greater anti-tumor activity and clinicalbenefit in such animals compared to those treated with the compound oranticancer drugs/radiation alone. Put another way, because the compoundsprovided herein can lower the apoptotic threshold of cells that expressp53 or p53-related protein, the proportion of cells that successfullyexecute the apoptosis program in response to the apoptosis inducingactivity of anticancer drugs/radiation will be increased when used incombination with one or more of the compounds provided herein.Alternatively, the compounds provided herein can be used to allowadministration of a lower, and therefore less toxic and more tolerable,dose of an anticancer drug and/or radiation to produce the same tumorresponse/clinical benefit as the conventional dose of the anticancerdrug/radiation alone. Since the doses for all approved anticancer drugsand radiation treatments are known, the present compounds, compositions,and methods provided herein can be used with one or more approvedanticancer drugs and/or radiation treatment. Also, since the compoundsprovided herein may act at least in part by stimulating thepro-apoptotic and/or cell cycle-inhibiting activities of p53 andp53-related proteins, the exposure of cancer cells and supporting cellsto therapeutically effective amounts of the compounds can be temporallylinked to coincide with the attempts of cells to execute the apoptosisprogram in response to the anticancer drug or radiation therapy. Thus,in some embodiments, administering the compounds or compositionsprovided herein in combination with other known anticancer drugs provideespecially efficacious therapeutic practices.

In other embodiments, the inhibitors of the interaction between p53 orp53-related proteins and MDM2 and MDM2-related proteins provided hereinmay protect normal (e.g., non-hyperproliferative) cells from the toxiceffects of certain chemotherapeutic agents and radiation, possiblythrough the ability of the inhibitors to induce cell cycle arrest ofnormal cells. For example, the inhibitors provided herein may cause cellcycle arrest in cells comprising wild-type or functional p53 (and/orwild-type or functional p53-related proteins) while having no or lesseffect on cancer cells comprising mutated, deleted, or otherwise non- orless functional p53 (and/or mutated, deleted, or otherwise non- or lessfunctional p53-related proteins). This differential protective effectmay allow for more effective treatment of cancer by allowing the use ofhigher doses or longer treatments of chemotherapeutic agents ortreatments without increasing the toxic side effects of such treatmentwhen administered in combination with inhibitors provided herein.

Applicants have found that certain spiro-oxindoles provided hereindisplay an unexpected combination of drug-like properties. Theunexpected combinations include, e.g., two or more of in vitro efficacy,in vivo efficacy, in vitro liver microsome stability, desirableabsorption, distribution, metabolism, and excretion (ADME) properties.For example, certain spiro-oxindoles provided herein are more resistantto metabolic degradation e.g., as measured by in vitro liver microsomalstability and/or in vivo pharmacokinetics, and/or display improved invivo efficacy as compared to known antagonists of the p53-MDM2interaction.

Applicants have also found that metabolically cleavable groups can beused to increase the aqueous solubility of the parent molecule. Thus, insome embodiments, the spiro-oxindoles provided herein are usefulprodrugs with improved aqueous solubility relative to the parentmolecule.

In some embodiments, spiro-oxindoles provided herein have Formula I:

wherein:

R^(1a), R^(1b), R^(1c), and R^(1d) are independently selected from thegroup consisting of hydrogen, halogen, hydroxy, amino, nitro, cyano,alkoxy, aryloxy, optionally substituted alkyl, haloalkyl, optionallysubstituted cycloalkyl, optionally substituted alkenyl, optionallysubstituted cycloalkenyl, optionally substituted aryl, optionallysubstituted heteroaryl, carboxamido, and sulfonamido;

R² is selected from the group consisting of optionally substituted aryland optionally substituted heteroaryl;

R³ is selected from the group consisting of optionally substitutedalkyl, optionally substituted (cycloalkyl)alkyl, optionally substitutedcycloalkyl, optionally substituted alkenyl, optionally substitutedcycloalkenyl, optionally substituted aryl, and optionally substitutedheteroaryl;

R⁴ is selected from the group consisting of hydrogen and optionallysubstituted C₁-C₆ alkyl;

R⁵ is selected from the group consisting of:

wherein:

each R^(6a) and R^(6b) is independently selected from the groupconsisting of hydrogen and optionally substituted C₁-C₆ alkyl;

R⁷ is selected from the group consisting of hydrogen, optionallysubstituted C₁-C₆ alkyl, and optionally substituted cycloalkyl;

R^(8a) and R^(8b) are each independently selected from the groupconsisting of hydrogen, optionally substituted C₁-C₆ alkyl, andoptionally substituted cycloalkyl; or

R^(8a) and R^(8b) taken together with the carbon that they are attachedform a 3- to 8-membered optionally substituted cycloalkyl;

W¹ is selected from the group consisting of −OR^(9a) and —NR^(9b)R^(9c);

R^(9a) is hydrogen;

R^(9b) is selected from the group consisting of hydrogen, optionallysubstituted alkyl, optionally substituted cycloalkyl, optionallysubstituted aryl, optionally substituted heteroaryl, —SO₂R^(9d), and—CONR^(9e)R^(9f);

R^(9c) is selected from the group consisting of hydrogen, optionallysubstituted alkyl, optionally substituted cycloalkyl, optionallysubstituted aryl, and optionally substituted heteroaryl; or

R^(9b) and R^(9c) taken together with the nitrogen atom to which theyare attached form a 4- to 8-membered optionally substituted heterocyclo;

R^(9d) is selected from the group consisting of optionally substitutedalkyl and optionally substituted cycloalkyl;

R^(9e) and R^(9f) are each independently selected from the groupconsisting of hydrogen, optionally substituted alkyl, and optionallysubstituted cycloalkyl; or

R^(9e) and R^(9f) taken together with the nitrogen atom to which theyare attached form a 4- to 8-membered optionally substituted heterocyclo;

W² is selected from the group consisting of —OR¹⁰ and —NR^(11a)R^(11b);

with the proviso that when W¹ is −OR^(9a) and W² is —OR¹⁰ then at leastone of R⁷, R^(8a), and R^(8b) is other than hydrogen;

R¹⁰ is hydrogen; or

one of R^(9a) and R¹⁰ is hydrogen and the other is a metabolicallycleavable group;

R^(11a) is selected from the group consisting of hydrogen, optionallysubstituted alkyl, optionally substituted cycloalkyl, optionallysubstituted aryl, optionally substituted heteroaryl, —SO₂R^(11c), and—CONR^(11d)R^(11e);

R^(11b) is selected from the group consisting of hydrogen, optionallysubstituted alkyl, optionally substituted cycloalkyl, optionallysubstituted aryl, and optionally substituted heteroaryl; or

R^(11a) and R^(11b) taken together with the nitrogen atom to which theyare attached form a 4- to 8-membered optionally substituted heterocyclo;

R^(11c) is selected from the group consisting of optionally substitutedalkyl and optionally substituted cycloalkyl;

R^(11d) and R^(11e) are each independently selected from the groupconsisting of hydrogen, optionally substituted alkyl, and optionallysubstituted cycloalkyl; or

R¹¹ and R^(11e) taken together with the nitrogen atom to which they areattached form a 4- to 8-membered optionally substituted heterocyclo;

n is 1, 2, 3, 4, or 5;

each R^(12a), R^(12b), R^(12c) and R^(12d) is independently selectedfrom the group consisting of hydrogen and optionally substituted C₁-C₆alkyl;

R¹³ is selected from the group consisting of hydrogen and optionallysubstituted C₁-C₆ alkyl;

R¹⁴ is selected from the group consisting of hydrogen, optionallysubstituted C₁-C₆ alkyl, and optionally substituted cycloalkyl;

Z is selected from the group consisting of —OR¹⁵ and —NR^(16a)R^(16b);or

Z and R¹⁴ taken together form a carbonyl, i.e., a C≡O, group.

R¹⁵ is selected from the group consisting of hydrogen and metabolicallycleavable group;

R^(16a) is selected from the group consisting of —SO₂R^(16c) and—CONR^(16d)R^(16e);

R^(16b) is selected from the group consisting of hydrogen and optionallysubstituted alkyl;

R^(16c) is selected from the group consisting of optionally substitutedalkyl, optionally substituted cycloalkyl, optionally substituted aryl,and optionally substituted heteroaryl;

R^(16d) and R^(16e) are each independently selected from the groupconsisting of hydrogen, optionally substituted alkyl, optionallysubstituted cycloalkyl, optionally substituted aryl, and optionallysubstituted heteroaryl; or

R^(16d) and R^(16e) taken together with the nitrogen atom to which theyare attached form a 4- to 8-membered heterocyclo;

o is 1, 2, or 3;

p is 0, 1, 2, or 3;

each R^(17a), R^(17b), R^(17c) and R^(17d) is independently selectedfrom the group consisting of hydrogen and optionally substituted C₁-C₆alkyl;

R¹⁸ is selected from the group consisting of hydrogen and optionallysubstituted C₁-C₆ alkyl;

R¹⁹ is selected from the group consisting of hydrogen, optionallysubstituted C₁-C₆ alkyl, and optionally substituted cycloalkyl;

R²⁰ is selected from the group consisting of hydrogen, optionallysubstituted C₁-C₆ alkyl, and optionally substituted cycloalkyl;

R^(21a) and R^(21b) are each hydrogen; or

one of R^(21a) and R^(21b) is hydrogen and the other is metabolicallycleavable group;

q is 0, 1, 2, or 3;

r is 1, 2, or 3;

each R^(22a), R^(22b), R^(22c), and R^(22d) is independently selectedfrom the group consisting of hydrogen and optionally substituted C₁-C₆alkyl;

R²³ is selected from the group consisting of hydrogen and optionallysubstituted C₁-C₆ alkyl;

R²⁴ is selected from the group consisting of —SO₂R^(24a) and—CONR^(24b)R^(24c);

R^(24a) is selected from the group consisting of optionally substitutedalkyl, optionally substituted cycloalkyl, optionally substituted aryl,and optionally substituted heteroaryl;

R^(24b) and R^(24c) are each independently selected from the groupconsisting of hydrogen, optionally substituted cycloalkyl, optionallysubstituted aryl, and optionally substituted heteroaryl; or

R^(24b) and R^(24c) taken together with the nitrogen atom to which theyare attached form a 4- to 8-membered heterocyclo;

s and t are each independently 1, 2, or 3;

X is selected from the group consisting of O, S, and NR′;

Y is selected from the group consisting of O, S, and NR″;

R′ is selected from the group consisting of hydrogen, optionallysubstituted alkyl, aralkyl, and optionally substituted cycloalkyl;

R″ is selected from the group consisting of hydrogen, optionallysubstituted alkyl, aralkyl, and optionally substituted cycloalkyl; and

represents a single or a double bond,

or a pharmaceutically acceptable salt, solvate, or prodrug thereof, or acompound having the structure:

or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

In some embodiments, the compounds provided herein inhibit theinteraction between p53 or p53-related proteins and MDM2 or MDM2-relatedproteins.

In some embodiments, the compounds provided herein contain ametabolically cleavable group. In particular, in some embodiments, thecompounds provided herein contain a hydroxy group of a hydroxycycloalkylside chain that can be used to attach a metabolically cleavable group.Suitable metabolically cleavable groups include, but are not limited to,amino acid esters or phosphate esters.

In some embodiments, the compounds provided herein can be used to inducesenescence, cell cycle arrest and/or apoptosis in cells containingfunctional p53 or p53-related proteins. Also provided herein are methodsof using the compounds provided herein for sensitizing cells toadditional agent(s), such as inducers of senescence, apoptosis and/orcell cycle arrest. The compounds provided herein can also be used toprovide chemoprotection of normal cells through the induction of cellcycle arrest prior to treatment with chemotherapeutic agents. In oneembodiment, the methods of rendering a normal cell resistant tochemotherapeutic agents or treatments comprises contacting the cell withone or more compounds provided herein. In one embodiment, methods ofprotecting normal cells in an animal having a hyperproliferative diseasefrom the toxic side effects of chemotherapeutic agents or treatments,comprises administering to the animal a compound provided herein.Provided herein are methods for the treatment, amelioration, orprevention of disorders, side effects, or conditions caused by theadministration of chemotherapeutic agents to normal cells comprisingadministering to an animal undergoing chemotherapy a compound providedherein. Examples of such disorders and conditions caused by chemotherapyinclude, without limitation, mucositis, stomatitis, xerostomia,gastrointestinal disorders, and alopecia.

The compounds provided herein are useful for the treatment,amelioration, or prevention of disorders, such as those responsive toinduction of apoptotic cell death, e.g., disorders characterized bydysregulation of apoptosis, including hyperproliferative diseases suchas cancer. In certain embodiments, the compounds can be used to treat,ameliorate, or prevent cancer that is characterized by resistance tocancer therapies (e.g., those cancer cells which are chemoresistant,radiation resistant, hormone resistant, and the like). In otherembodiments, the compounds can be used to treat hyperproliferativediseases characterized by expression of functional p53 or p53-relatedproteins. In other embodiments, the compounds provided herein can beused to protect normal (e.g., non-hyperproliferative) cells from thetoxic side effects of chemotherapeutic agents and treatments by theinduction of cell cycle arrest in those cells.

In one embodiment, pharmaceutical compositions are provided. Thepharmaceutical compositions can comprise one of more of the compoundsprovided herein in a pharmaceutically acceptable carrier.

In one embodiment, kits are provided. The kits can comprise one or moreof the compounds provided herein and instructions for administering thecompound to an animal. The kits may optionally contain other therapeuticagents, e.g., anticancer agents or apoptosis-modulating agents.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a bar graph showing the induction of cell death by MDM2inhibitors in SJSA-1 cancer cells with wild-type p53.

FIG. 2 is an illustration showing western blot analysis of p53activation induced by MDM2 inhibitors in SJSA-1 cancer cells withwild-type p53.

FIG. 3 is an illustration showing western blot analysis of p53activation induced by MDM2 inhibitors in SJSA-1 cancer cells withwild-type p53.

FIG. 4 is an illustration showing western blot analysis of p53activation induced by MDM2 inhibitors in SJSA-1 cancer cells withwild-type p53.

FIG. 5 is a line graph showing the plasma concentration vs. time curvesfollowing intravenous (IV) dosing of MDM2 inhibitors in rat.

FIG. 6 is a line graph showing the plasma concentration vs. time curvesfollowing oral dosing of MDM2 inhibitors in rat.

FIG. 7 is a line graph showing in vivo antitumor activity of MDM2inhibitors in the SJSA-1 xenograft model in mice.

FIG. 8 is a line graph showing the animal weight followingadministration of MDM2 inhibitors in mice.

FIG. 9 is a line graph showing in vivo antitumor activity of MDM2inhibitors in the SJSA-1 xenograft model in mice.

FIG. 10 is a line graph showing the animal weight followingadministration of MDM2 inhibitors in mice.

FIG. 11 is a line graph showing in vivo antitumor activity of MDM2inhibitors in the SJSA-1 xenograft model in mice.

FIG. 12 is a line graph showing the animal weight followingadministration of MDM2 inhibitors in mice.

FIG. 13A-D are four MS/MS spectra of protonated MI-219, MI-142, MI-63and MI-708B, respectively.

FIG. 14A-B are two MS/MS spectra of deprotonated MI-219 and MI-142,respectively.

FIG. 15A-D are four LC-MS chromatograms and MS/MS spectra of M1 in ratplasma (A and B) and synthetic M1 (C and D).

FIG. 16 is a line graph showing plasma concentrations of MI-219 and M1in the rats.

FIG. 17A-B are two MS/MS spectra of protonated M2 (A) and deprotonatedM2 (B).

FIG. 18A-D are four MS/MS spectra of protonated MI-773 and threemetabolites M1, M2 and M3.

FIG. 19A-C are three MS/MS spectra of protonated MI-519-63 and itsmetabolites M1 and M2.

FIG. 20 is a reverse phase HPLC chromatogram of MI-519-64 afterisolation by column chromatography on silica gel.

FIG. 21 is reverse phase HPLC chromatogram of MI-519-64 after treatmentwith acetonitrile/water for 12 h. Three isomers are present. MI-519-64and MI-519-6401 correspond to RP-HPLC peaks at 30.578 minutes, and31.787 minutes, respectively. The isomer eluting at 29.162 minutes hasnot been fully characterized (referred to as MI-519-6402).

FIG. 22 is reverse phase HPLC chromatogram of MI-519-64 after treatmentwith acetonitrile/water for 3 days.

FIG. 23 is a line graph showing the binding affinities of MI-519-64,MI-519-6401, and MI-519-6402 to human MDM2 protein, as determined usinga fluorescence-polarization binding assay. The purity of each isomerused in this experiment (as determined by RP-HPLC) are as follows:MI-519-6402: 90% (with 10% of MI-519-64); MI-519-64: 93% (with 3% ofMI-519-6402 and 4% of MI-519-6401); and MI-519-6401: >99%. The log EC₅₀values for MI-519-6402, MI-519-64, and MI-519-6401 are 2.030 nM, 1.598nM, and 0.8354 nM, respectively.

FIG. 24 is a line graph show the binding affinities of MI-773 andMI-77301 to human MDM2 protein, as determined using afluorescence-polarization binding assay.

FIG. 25 is a line graph showing the stability of MI-773 (TFA salt) atvarious time points. The compounds corresponding to peaks 1 and 2 havenot been fully characterized. The compound corresponding to peak 3 isMI-77301.

FIG. 26 is a line graph showing the stability of MI-77301 (TFA salt) atvarious time points.

FIG. 27 is an illustration showing western blot analysis of p53activation and apoptosis induced by MI-773 and MI-77301 in the SJSA-1cell line.

FIG. 28 is an illustration showing western blot analysis of p53activation and apoptosis induced by MI-519-64 and MI-519-6401 in theSJSA-1 cell line.

FIG. 29 is a bar graph showing apoptosis induced by MI-773 and MI-77301in the SJSA-1 cell line.

FIG. 30 is a bar graph showing cell death induced by MI-519-64 andMI-519-6401 in the SJSA-1 cell line.

FIG. 31 is a bar graph showing cell death induced by MI-519-64 andMI-519-6401 in the RS4; 11 cell line.

FIG. 32 is an illustration showing western blot analysis of in vivoactivation of p53 and PARP cleavage induced by MI-519-64 and MI-519-6401in SJSA-1 tumors in mice.

FIG. 33 is an illustration showing western blot analysis of in vivoactivation of p53 and PARP cleavage induced by MI-519-64 and MI-519-6401in RS4; 11 tumors in mice.

FIG. 34 is an illustration showing western blot analysis of in vivoactivation of p53 and PARP cleavage induced by MI-773 and MI-77301 inSJSA-1 tumors in mice.

FIG. 35 is an illustration showing three western blot analyses of p53activation and apoptosis induced by MI-773 and MI-77301 in the RS4; 11cell line.

FIG. 36 is a line graph showing in vivo antitumor activity of MI-519-64,MI-519-6401, MI-773, and MI-77301 in the SJSA-1 xenograft model in mice.

FIG. 37 is a line graph showing the animal weight followingadministration of MI-519-64, MI-519-6401, MI-773, and MI-77301 in mice.

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are compounds that inhibit the interaction between p53or p53-related proteins and MDM2 or MDM2-related proteins. By inhibitingthe negative effect of MDM2 or MDM2-related proteins on p53 orp53-related proteins, these compounds sensitize cells to inducers ofapoptosis and/or cell cycle arrest. In some embodiments, the compoundsprovided herein induce apoptosis and/or cell cycle arrest. Therefore,also provided herein are methods of sensitizing cells to inducers ofapoptosis and/or cell cycle arrest and to methods of inducing apoptosisand/or cell cycle arrest in cells. In some embodiments, the methodscomprise contacting the cells with one or more compounds provided hereinalone or in combination with additional agent(s), e.g., an inducer ofapoptosis or a cell cycle disrupter.

Also provided herein are methods of treating, ameliorating, orpreventing disorders in an patient, comprising administering to thepatient one or more compounds provided herein and additional agent(s),e.g., an inducer of apoptosis. Such disorders include thosecharacterized by a dysregulation of apoptosis and those characterized bythe proliferation of cells expressing functional p53 or p53-relatedproteins. In other embodiments, methods of protecting normal (e.g.,non-hyperproliferative) cells in an animal from the toxic side effectsof chemotherapeutic agents and treatments are provided. The methodscomprise administering to the animal one or more compounds providedherein.

DEFINITIONS

The term “anticancer agent” as used herein, refers to any therapeuticagent (e.g., chemotherapeutic compound and/or molecular therapeuticcompound), antisense therapy, radiation therapy, or surgicalintervention, used in the treatment of hyperproliferative diseases suchas cancer (e.g., in mammals, e.g., in humans).

The term “prodrug” as used herein, refers to a pharmacologicallyinactive derivative of a parent “drug” molecule that requiresbiotransformation (e.g., either spontaneous or enzymatic) within thetarget physiological system to release, or to convert (e.g.,enzymatically, physiologically, mechanically, electromagnetically) theprodrug into the active drug. Prodrugs are designed to overcome problemsassociated with stability, water solubility, toxicity, lack ofspecificity, or limited bioavailability. Exemplary prodrugs comprise anactive drug molecule itself and a chemical masking group (e.g., a groupthat reversibly suppresses the activity of the drug). Some prodrugs arevariations or derivatives of compounds that have groups cleavable undermetabolic conditions. Prodrugs can be readily prepared from the parentcompounds using methods known in the art, such as those described in ATextbook of Drug Design and Development, Krogsgaard-Larsen and H.Bundgaard (eds.), Gordon & Breach, 1991, particularly Chapter 5: “Designand Applications of Prodrugs”; Design of Prodrugs, H. Bundgaard (ed.),Elsevier, 1985; Prodrugs: Topical and Ocular Drug Delivery, K. B. Sloan(ed.), Marcel Dekker, 1998; Methods in Enzymology, K. Widder et al.(eds.), Vol. 42, Academic Press, 1985, particularly pp. 309-396;Burger's Medicinal Chemistry and Drug Discovery, 5th Ed., M. Wolff(ed.), John Wiley & Sons, 1995, particularly Vol. 1 and pp. 172-178 andpp. 949-982; Pro-Drugs as Novel Delivery Systems, T. Higuchi and V.Stella (eds.), Am. Chem. Soc., 1975; and Bioreversible Carriers in DrugDesign, E. B. Roche (ed.), Elsevier, 1987.

Exemplary prodrugs become pharmaceutically active in vivo or in vitrowhen they undergo solvolysis under physiological conditions or undergoenzymatic degradation or other biochemical transformation (e.g.,phosphorylation, hydrogenation, dehydrogenation, glycosylation).Prodrugs often offer advantages of water solubility, tissuecompatibility, or delayed release in the mammalian organism. (See e.g.,Bundgard, Design of Prodrugs, pp. 7-9, 21-24, Elsevier, Amsterdam(1985); and Silverman, The Organic Chemistry of Drug Design and DrugAction, pp. 352-401, Academic Press, San Diego, Calif. (1992)). Commonprodrugs include acid derivatives such as esters prepared by reaction ofparent acids with a suitable alcohol (e.g., a lower alkanol) or estersprepared by reaction of parent alcohol with a suitable carboxylic acid,(e.g., an amino acid), amides prepared by reaction of the parent acidcompound with an amine, basic groups reacted to form an acylated basederivative (e.g., a lower alkylamide), or phosphorus-containingderivatives, e.g., phosphate, phosphonate, and phosphoramidate esters,including cyclic phosphate, phosphonate, and phosphoramidate, see, e.g.,US 2007/0249564 A1.

The term “metabolically cleavable group” as used herein, refers togroups which can be cleaved from the parent molecule by metabolicprocesses and be substituted with hydrogen. Certain compounds containingmetabolically cleavable groups may be prodrugs, i.e., they arepharmacologically inactive. Certain other compounds containingmetabolically cleavable groups may be antagonists of the interactionbetween p53 and MDM2. In such cases, these compounds may have more,less, or equivalent activity of the parent molecule. Examples ofmetabolically cleavable groups include those derived from amino acids(see, e.g., US 2006/0241017 A1; US 2006/0287244 A1; and WO 2005/046575A2) or phosphorus-containing compounds (see, e.g., U.S. 2007/0249564 A1)as illustrated in Scheme 1.

The term “pharmaceutically acceptable salt” as used herein, refers toany salt (e.g., obtained by reaction with an acid or a base) of acompound provided herein that is physiologically tolerated in the targetanimal (e.g., a mammal). Salts of the compounds of provided herein maybe derived from inorganic or organic acids and bases. Examples of acidsinclude, but are not limited to, hydrochloric, hydrobromic, sulfuric,nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic,salicylic, succinic, toluene-p-sulfonic, tartaric, acetic, citric,methanesulfonic, ethanesulfonic, formic, benzoic, malonic, sulfonic,naphthalene-2-sulfonic, benzenesulfonic acid, and the like. Other acids,such as oxalic, while not in themselves pharmaceutically acceptable, maybe employed in the preparation of salts useful as intermediates inobtaining the compounds provided herein and their pharmaceuticallyacceptable acid addition salts.

Examples of bases include, but are not limited to, alkali metal (e.g.,sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides,ammonia, and compounds of formula NW₄ ⁺ wherein W is C₁₋₄ alkyl, and thelike.

Examples of salts include, but are not limited to: acetate, adipate,alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate,citrate, camphorate, camphorsulfonate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, flucoheptanoate,glycerophosphate, hemisulfate, heptanoate, hexanoate, chloride, bromide,iodide, 2-hydroxyethanesulfonate, lactate, maleate, mesylate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmoate,pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate,succinate, tartrate, thiocyanate, tosylate, undecanoate, and the like.Other examples of salts include anions of the compounds provided hereincompounded with a suitable cation such as Na⁺, NH₄ ⁺, and NW₄ ⁺ (whereinW is a C₁₋₄ alkyl group), and the like. For therapeutic use, salts ofthe compounds provided herein are contemplated as being pharmaceuticallyacceptable. However, salts of acids and bases that arenon-pharmaceutically acceptable may also find use, for example, in thepreparation or purification of a pharmaceutically acceptable compound.

The term “solvate” as used herein, refers to the physical association ofa compound provided herein with one or more solvent molecules, whetherorganic or inorganic. This physical association often includes hydrogenbonding. In certain instances, the solvate is capable of isolation, forexample, when one or more solvate molecules are incorporated in thecrystal lattice of the crystalline solid. “Solvate” encompasses bothsolution-phase and isolable solvates. Exemplary solvates includehydrates, ethanolates, and methanolates.

The term “monovalent pharmaceutically acceptable cation” as used hereinrefers to inorganic cations such as, but not limited to, alkaline metalions, e.g., Na⁺ and K⁺, as well as organic cations such as, but notlimited to, ammonium and substituted ammonium ions, e.g., NH₄ ⁺, NHMe₃⁺, NH₂Me₂ ⁺, NHMe₃ ⁺ and NMe₄ ⁺.

The term “divalent pharmaceutically acceptable cation” as used hereinrefers to inorganic cations such as, but not limited to, alkaline earthmetal cations, e.g., Ca²⁺ and Mg²⁺.

Examples of monovalent and divalent pharmaceutically acceptable cationsare discussed, e.g., in Berge et al. J. Pharm. Sci., 66:1-19 (1997).

The term “therapeutically effective amount,” as used herein, refers tothat amount of the therapeutic agent (including the compounds andcompositions of matter provided herein) sufficient to result inamelioration of one or more symptoms of a disorder, or preventadvancement of a disorder, or cause regression of the disorder. Forexample, with respect to the treatment of cancer, in one embodiment, atherapeutically effective amount can refer to the amount of atherapeutic agent that decreases the rate of tumor growth, decreasestumor mass, decreases the number of metastases, increases time to tumorprogression, increase tumor cell apoptosis, or increases survival timeby at least 5%, at least 10%, at least 15%, at least 20%, at least 25%,at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or at least 100%.

The terms “sensitize” and “sensitizing,” as used herein, refer tomaking, through the administration of a first therapeutic agent (e.g., acompound provided herein), an animal or a cell within an animal moresusceptible, or more responsive, to the biological effects (e.g.,promotion or retardation of an aspect of cellular function including,but not limited to, cell division, cell growth, proliferation, invasion,angiogenesis, necrosis, or apoptosis) of a second therapeutic agent. Thesensitizing effect of a first agent on a target cell can be measured asthe difference in the intended biological effect (e.g., promotion orretardation of an aspect of cellular function including, but not limitedto, cell growth, proliferation, invasion, angiogenesis, or apoptosis)observed upon the administration of a second agent with and withoutadministration of the first agent. The response of the sensitized cellcan be increased by at least about 10%, at least about 20%, at leastabout 30%, at least about 40%, at least about 50%, at least about 60%,at least about 70%, at least about 80%, at least about 90%, at leastabout 100%, at least about 150%, at least about 200%, at least about250%, at least 300%, at least about 350%, at least about 400%, at leastabout 450%, or at least about 500% over the response in the absence ofthe first agent.

The term “dysregulation of apoptosis,” as used herein, refers to anyaberration in the ability of (e.g., predisposition) a cell to undergocell death via apoptosis. Dysregulation of apoptosis is associated withor induced by a variety of conditions, non-limiting examples of whichinclude, autoimmune disorders (e.g., systemic lupus erythematosus,rheumatoid arthritis, graft-versus-host disease, myasthenia gravis, orSjögren's syndrome), chronic inflammatory conditions (e.g., psoriasis,asthma or Crohn's disease), hyperproliferative disorders (e.g., tumors,B cell lymphomas, or T cell lymphomas), viral infections (e.g., herpes,papilloma, or HIV), and other conditions such as osteoarthritis andatherosclerosis. It should be noted that when the dysregulation isinduced by or associated with a viral infection, the viral infection mayor may not be detectable at the time dysregulation occurs or isobserved. That is, viral-induced dysregulation can occur even after thedisappearance of symptoms of viral infection.

The term “functional p53,” as used herein, refers to wild-type p53expressed at normal, high, or low levels and mutant or allelic variantsof p53 that retain(s) at least about 5% of the activity of wild-typep53, e.g., at least about 10%, about 20%, about 30%, about 40%, about50%, or more of wild-type activity.

The term “p53-related protein,” as used herein, refers to proteins thathave at least 25% sequence homology with p53, have tumor suppressoractivity, and are inhibited by interaction with MDM2 or MDM2-relatedproteins. Examples of p53-related proteins include, but are not limitedto, p63 and p73.

The term “MDM2-related protein,” as used herein, refers to proteins thathave at least 25% sequence homology with MDM2, and interact with andinhibit p53 or p53-related proteins. Examples of MDM2-related proteinsinclude, but are not limited to, MDMX. The term “senescence” as usedherein, refers to the phenomenon whereby non-cancerous diploid cellslose the ability to divide, and characterized in part by telomericdysfunction or shortening.

The term “hyperproliferative disease,” as used herein, refers to anycondition in which a localized population of proliferating cells in ananimal is not governed by the usual limitations of normal growth.Examples of hyperproliferative disorders include tumors, neoplasms,lymphomas, leukemias and the like. A neoplasm is said to be benign if itdoes not undergo invasion or metastasis and malignant if it does eitherof these. A “metastatic” cell means that the cell can invade neighboringbody structures. Hyperplasia is a form of cell proliferation involvingan increase in cell number in a tissue or organ without significantalteration in structure or function. Metaplasia is a form of controlledcell growth in which one type of fully differentiated cell substitutesfor another type of differentiated cell.

The pathological growth of activated lymphoid cells often results in anautoimmune disorder or a chronic inflammatory condition. As used herein,the term “autoimmune disorder” refers to any condition in which anorganism produces antibodies or immune cells which recognize theorganism's own molecules, cells or tissues. Non-limiting examples ofautoimmune disorders include autoimmune hemolytic anemia, autoimmunehepatitis, Berger's disease or IgA nephropathy, celiac sprue, chronicfatigue syndrome, Crohn's disease, dermatomyositis, fibromyalgia, graftversus host disease, Grave's disease, Hashimoto's thyroiditis,idiopathic thrombocytopenia purpura, lichen planus, multiple sclerosis,myasthenia gravis, psoriasis, rheumatic fever, rheumatic arthritis,scleroderma, Sjögren's syndrome, systemic lupus erythematosus, type 1diabetes, ulcerative colitis, vitiligo, and the like.

The term “neoplastic disease,” as used herein, refers to any abnormalgrowth of cells being either benign (non-cancerous) or malignant(cancerous).

The term “normal cell,” as used herein, refers to a cell that is notundergoing abnormal growth or division. Normal cells are non-cancerousand are not part of any hyperproliferative disease or disorder.

The term “anti-neoplastic agent,” as used herein, refers to any compoundthat retards the proliferation, growth, or spread of a targeted (e.g.,malignant) neoplasm.

The terms “prevent,” “preventing,” and “prevention,” as used herein,refer to a decrease in the occurrence of pathological cells (e.g.,hyperproliferative or neoplastic cells) in an animal. The prevention maybe complete, e.g., the total absence of pathological cells in a subject.The prevention may also be partial, such that the occurrence ofpathological cells in a subject is less than that which would haveoccurred without treatment with one or more compounds provided herein.

The term “apoptosis-modulating agents,” as used herein, refers to agentswhich are involved in modulating (e.g., inhibiting, decreasing,increasing, promoting) apoptosis. Examples of apoptosis-modulatingagents include proteins which comprise a death domain such as, but notlimited to, Fas/CD95, TRAMP, TNF RI, DR1, DR2, DR3, DR4, DR5, DR6, FADD,and RIP. Other examples of apoptosis-modulating agents include, but arenot limited to, TNFα, Fas ligand, antibodies to Fas/CD95 and other TNFfamily receptors, TRAIL (also known as Apo2 Ligand or Apo2L/TRAIL),antibodies to TRAIL-R1 or TRAIL-R2, Bcl-2, p53, BAX, BAD, Akt, CAD, PI3kinase, PP1, and caspase proteins. Modulating agents broadly includeagonists and antagonists of TNF family receptors and TNF family ligands.Apoptosis-modulating agents may be soluble or membrane bound (e.g.ligand or receptor). Apoptosis-modulating agents include those which areinducers of apoptosis, such as TNF or a TNF-related ligand, particularlya TRAMP ligand, a Fas/CD95 ligand, a TNFR-1 ligand, or TRAIL.

The term “pharmaceutically acceptable carrier” or “pharmaceuticallyacceptable vehicle” encompasses any of the standard pharmaceuticalcarriers, solvents, surfactants, or vehicles. Suitable pharmaceuticallyacceptable vehicles include aqueous vehicles and nonaqueous vehicles.Standard pharmaceutical carriers and their formulations are described inRemington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.,19th ed. 1995.

The term “alkyl” as used herein by itself or part of another grouprefers to a straight-chain or branched saturated aliphatic hydrocarbonhaving from one to eighteen carbons or the number of carbons designated(e.g., C₁-C₁₈ means 1 to 18 carbons). In one embodiment, the alkyl is aC₁-C₁₀ alkyl. In another embodiment, the alkyl is a C₁-C₆ alkyl. Inanother embodiment, the alkyl is a C₁-C₄ alkyl. Exemplary alkyl groupsinclude methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl,isobutyl, tert-butyl, n-pentyl, n-hexyl, isohexyl, n-heptyl,4,4-dimethylpentyl, n-octyl, 2,2,4-trimethylpentyl, nonyl, decyl and thelike.

The term “optionally substituted alkyl” as used herein by itself or partof another group means that the alkyl as defined above is eitherunsubstituted or substituted with one, two or three substituentsindependently selected from hydroxy (i.e., —OH), nitro (i.e., —NO₂),cyano (i.e., —CN), optionally substituted cycloalkyl, optionallysubstituted heteroaryl, optionally substituted heterocyclo, alkoxy,aryloxy, aralkyloxy, alkylthio, carboxamido or sulfonamido. In oneembodiment, the optionally substituted alkyl is substituted with twosubstituents. In another embodiment, the optionally substituted alkyl issubstituted with one substituent. In another embodiment, thesubstituents are selected from hydroxyl (i.e., a hydroxyalkyl),optionally substituted cycloalkyl (i.e., a (cycloalkyl)alkyl), or amino(i.e., an aminoalkyl). Exemplary optionally substituted alkyl groupsinclude —CH₂OCH₃, —CH₂CH₂NH₂, —CH₂CH₂NH(CH₃), —CH₂CH₂CN, —CH₂SO₂CH₃,hydroxymethyl, hydroxyethyl, hydroxypropyl, and the like.

The term “alkylenyl” as used herein by itself or part of another grouprefers to a divalent alkyl radical containing one, two, three, four, ormore joined methylene groups. Exemplary alkylenyl groups include—(CH₂)—, —(CH₂)₂—, —(CH₂)₃—, —(CH₂)₄—, and the like.

The term “optionally substituted alkylenyl” as used herein by itself orpart of another group means the alkylenyl as defined above is eitherunsubstituted or substituted with one, two, three, or four substituentsindependently selected from the group consisting of optionallysubstituted C₁-C₆ alkyl, optionally substituted cycloalkyl, optionallysubstituted aryl, and optionally substituted heteroaryl. In oneembodiment, the optionally substituted C₁-C₆ alkyl is methyl. In oneembodiment, the optionally substituted aryl is a phenyl optionallysubstituted with one or two halo groups. Exemplary optionallysubstituted alkylenyl groups include —CH(CH₃)—, —C(CH₃)₂—, —CH₂CH(CH₃)—,—CH₂CH(CH₃)CH₂—, —CH₂CH(Ph)CH₂—, —CH(CH₃)CH(CH₃)—, and the like.

The term “haloalkyl” as used herein by itself or part of another grouprefers to an alkyl as defined above having one to six halo substituents.In one embodiment, the haloalkyl has one, two or three halosubstituents. Exemplary haloalkyl groups include trifluoromethyl,—CH₂CH₂F and the like.

The term “hydroxyalkyl” as used herein by itself or part of anothergroup refers to an alkyl as defined above having one hydroxysubstituent. Exemplary hydroxyalkyl groups include hydroxymethyl,hydroxyethyl, hydroxypropyl, and the like.

The term “dihydroxyalkyl” as used herein by itself or part of anothergroup refers to alkyl as defined above having two hydroxyl substituents.Exemplary dihydroxyalkyl groups include —CH₂CH₂CCH₃(OH)CH₂OH,—CH₂CH₂CH(OH)CH(CH₃)OH, —CH₂CH(CH₂OH)₂,—CH₂CH₂CH(OH)C(CH₃)₂OH—CH₂CH₂CCH₃(OH)CH(CH₃)OH, and the like, includingstereoisomers thereof.

The term “hydroxycycloalkyl” as used herein by itself or part of anothergroup refers to an optionally substituted cycloalkyl as defined belowhaving a least one, e.g., one or two hydroxy substituents. Exemplaryhydroxycycloalkyl groups include:

and the like, including stereoisomers thereof.

The term “optionally substituted (cycloalkyl)alkyl” as used herein byitself or part of another group refers to an optionally substitutedalkyl as defined above having an optionally substituted cycloalkyl (asdefined below) substituent. Exemplary optionally substituted(cycloalkyl)alkyl groups include:

and the like, including stereoisomers thereof.

The term “aralkyl” as used herein by itself or part of another grouprefers to an optionally substituted alkyl as defined above having one,two or three optionally substituted aryl substituents. In oneembodiment, the aralkyl has two optionally substituted arylsubstituents. In another embodiment, the aralkyl has one optionallysubstituted aryl substituent. In another embodiment, the aralkyl is anaryl(C₁-C₄ alkyl). In another embodiment, the aryl(C₁-C₄ alkyl) has twooptionally substituted aryl substituents. In another embodiment, thearyl(C₁-C₄ alkyl) has one optionally substituted aryl substituent.Exemplary aralkyl groups include, for example, benzyl, phenylethyl,(4-fluorophenyl)ethyl, phenylpropyl, diphenylmethyl (i.e., Ph₂CH—),diphenylethyl (Ph₂CHCH₂—) and the like.

The term “cycloalkyl” as used herein by itself or part of another grouprefers to saturated and partially unsaturated (containing one or twodouble bonds) cyclic hydrocarbon groups containing one to three ringshaving from three to twelve carbon atoms (i.e., C₃-C₁₂ cycloalkyl) orthe number of carbons designated. In one embodiment, the cycloalkyl hasone ring. In another embodiment, the cycloalkyl is a C₃-C₆ cycloalkyl.Exemplary cycloalkyl groups include cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, norbornyl, decalin,adamantyl and the like.

The term “optionally substituted cycloalkyl” as used herein by itself orpart of another group means the cycloalkyl as defined above is eitherunsubstituted or substituted with one, two or three substituentsindependently selected from halo, nitro, cyano, hydroxy, amino,optionally substituted alkyl, haloalkyl, hydroxyalkyl, aminoalkyl,aralkyl, optionally substituted cycloalkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substituted aryl,optionally substituted heteroaryl, optionally substituted heterocyclo,alkoxy, aryloxy, aralkyloxy, alkylthio, carboxamido or sulfonamido. Theterm “optionally substituted cycloalkyl” also means the cycloalkyl asdefined above may be fused to an optionally substituted aryl. Exemplaryoptionally substituted cycloalkyl groups include

and the like.

The term “alkenyl” as used herein by itself or part of another grouprefers to an alkyl group as defined above containing one, two or threecarbon-to-carbon double bonds. In one embodiment, the alkenyl has onecarbon-to-carbon double bond. Exemplary alkenyl groups include —CH═CH₂,—CH₂CH═CH₂, —CH₂CH₂CH═CH₂, —CH₂CH₂CH═CHCH₃ and the like.

The term “optionally substituted alkenyl” as used herein by itself orpart of another group means the alkenyl as defined above is eitherunsubstituted or substituted with one, two or three substituentsindependently selected from halo, nitro, cyano, hydroxy, amino,optionally substituted alkyl, haloalkyl, hydroxyalkyl, aralkyl,optionally substituted cycloalkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted heterocyclo, alkoxy,aryloxy, aralkyloxy, alkylthio, carboxamido or sulfonamido. Exemplaryoptionally substituted alkenyl groups include —CH═CHPh, —CH₂CH═CHPh andthe like.

The term “cycloalkenyl” as used herein by itself or part of anothergroup refers to a cycloalkyl group as defined above containing one, twoor three carbon-to-carbon double bonds. In one embodiment, thecycloalkenyl has one carbon-to-carbon double bond. Exemplarycycloalkenyl groups include cyclopentene, cyclohexene and the like.

The term “optionally substituted cycloalkenyl” as used herein by itselfor part of another group means the cycloalkenyl as defined above iseither unsubstituted or substituted with one, two or three substituentsindependently selected from halo, nitro, cyano, hydroxy, amino,optionally substituted alkyl, haloalkyl, hydroxyalkyl, aralkyl,optionally substituted cycloalkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted heterocyclo, alkoxy,aryloxy, aralkyloxy, alkylthio, carboxamido or sulfonamido.

The term “alkynyl” as used herein by itself or part of another grouprefers to an alkyl group as defined above containing one to threecarbon-to-carbon triple bonds. In one embodiment, the alkynyl has onecarbon-to-carbon triple bond. Exemplary alkynyl groups include —C≡CH,—C≡CCH₃, —CH₂C≡CH, —CH₂CH₂C≡CH and —CH₂CH₂C≡CCH₃.

The term “optionally substituted alkynyl” as used herein by itself orpart of another group means the alkynyl as defined above is eitherunsubstituted or substituted with one, two or three substituentsindependently selected from halo, nitro, cyano, hydroxy, amino,optionally substituted alkyl, haloalkyl, hydroxyalkyl, aralkyl,optionally substituted cycloalkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted heterocyclo, alkoxy,aryloxy, aralkyloxy, alkylthio, carboxamido or sulfonamido. Exemplaryoptionally substituted alkenyl groups include —C≡CPh, —CH₂C≡CPh and thelike.

The term “aryl” as used herein by itself or part of another group refersto monocyclic and bicyclic aromatic ring systems having from six tofourteen carbon atoms (i.e., C₆-C₁₄ aryl) such as phenyl (abbreviated asPh), 1-naphthyl and 2-naphthyl and the like.

The term “optionally substituted aryl” as used herein by itself or partof another group means the aryl as defined above is either unsubstitutedor substituted with one to five substituents independently selected fromhalo, nitro, cyano, hydroxy, amino, optionally substituted alkyl,haloalkyl, hydroxyalkyl, aralkyl, optionally substituted cycloalkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, optionally substituted heteroaryl,optionally substituted heterocyclo, alkoxy, aryloxy, aralkyloxy,alkylthio, carboxamido or sulfonamido. In one embodiment, the optionallysubstituted aryl is an optionally substituted phenyl. In one embodiment,the optionally substituted phenyl has four substituents. In anotherembodiment, the optionally substituted phenyl has three substituents. Inanother embodiment, the optionally substituted phenyl has twosubstituents. In another embodiment, the optionally substituted phenylhas one substituent. Exemplary substituted aryl groups include2-methylphenyl, 2-methoxyphenyl, 2-fluorophenyl, 2-chlorophenyl,2-bromophenyl, 3-methylphenyl, 3-methoxyphenyl, 3-fluorophenyl,3-chlorophenyl, 4-methylphenyl, 4-ethylphenyl, 4-methoxyphenyl,4-fluorophenyl, 4-chlorophenyl, 2,6-di-fluorophenyl,2,6-di-chlorophenyl, 2-methyl, 3-methoxyphenyl, 2-ethyl,3-methoxyphenyl, 3,4-di-methoxyphenyl, 3,5-di-fluorophenyl3,5-di-methylphenyl and 3,5-dimethoxy, 4-methylphenyl,2-fluoro-3-chlorophenyl, 3-chloro-4-fluorophenyl and the like. The termoptionally substituted aryl is meant to include groups having fusedoptionally substituted cycloalkyl and fused optionally substitutedheterocyclo rings. Examples include

and the like.

The term “heteroaryl” as used herein by itself or part of another grouprefers to monocyclic and bicyclic aromatic ring systems having from fiveto fourteen carbon atoms (i.e., C₅-C₁₄ heteroaryl) and one, two, threeor four heteroatoms independently selected from the group consisting ofoxygen, nitrogen and sulfur. In one embodiment, the heteroaryl has threeheteroatoms. In one embodiment, the heteroaryl has two heteroatoms. Inone embodiment, the heteroaryl has one heteroatom. Exemplary heteroarylgroups include 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 2-imidazolyl,4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl,3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl,5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl,3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, purinyl,2-benzimidazolyl, 4-benzimidazolyl, 5-benzimidazolyl, 2-benzthiazolyl,4-benzthiazolyl, 5-benzthiazolyl, 5-indolyl, 3-indazolyl, 4-indazolyl,5-indazolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl,5-quinoxalinyl, 2-quinolyl 3-quinolyl, 6-quinolyl and the like. The termheteroaryl is meant to include possible N-oxides. Exemplary N-oxidesinclude pyridyl N-oxide and the like.

The term “optionally substituted heteroaryl” as used herein by itself orpart of another group means the heteroaryl as defined above is eitherunsubstituted or substituted with one to four substituents, typicallyone or two substituents, independently selected from halo, nitro, cyano,hydroxy, amino, optionally substituted alkyl, haloalkyl, hydroxyalkyl,aralkyl, optionally substituted cycloalkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substituted aryl,optionally substituted heteroaryl, optionally substituted heterocyclo,alkoxy, aryloxy, aralkyloxy, alkylthio, carboxamido or sulfonamido. Inone embodiment, the optionally substituted heteroaryl has onesubstituent. In another embodiment, the substituent is an optionallysubstituted aryl, aralkyl, or optionally substituted alkyl. In anotherembodiment, the substituent is an optionally substituted phenyl. Anyavailable carbon or nitrogen atom may be substituted. Exemplaryoptionally substituted heteroaryl groups include

and the like.

The term “heterocyclo” as used herein by itself or part of another grouprefers to saturated and partially unsaturated (containing one or twodouble bonds) cyclic groups containing one to three rings having fromtwo to twelve carbon atoms (i.e., C₂-C₁₂ heterocyclo) and one or twooxygen, sulfur or nitrogen atoms. The heterocyclo can be optionallylinked to the rest of the molecule through a carbon or nitrogen atom.Exemplary heterocyclo groups include

and the like.

The term “optionally substituted heterocyclo” as used herein by itselfor part of another group means the heterocyclo as defined above iseither unsubstituted or substituted with one to four substituentsindependently selected from halo, nitro, cyano, hydroxy, amino,optionally substituted alkyl, haloalkyl, hydroxyalkyl, aralkyl,optionally substituted cycloalkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted heterocyclo, alkoxy,aryloxy, aralkyloxy, alkylthio, carboxamido, sulfonamido, —COR^(c),—SO₂R^(d), —N(R^(e))COR^(f), —N(R^(e))SO₂R^(g) or—N(R^(e))C═N(R^(h))-amino, wherein R^(c) is hydrogen, optionallysubstituted alkyl, optionally substituted aryl, or optionallysubstituted heteroaryl; R^(d) is optionally substituted alkyl,optionally substituted aryl, or optionally substituted heteroaryl; R^(e)is hydrogen, optionally substituted alkyl, optionally substituted aryl,or optionally substituted heteroaryl; R^(f) is hydrogen, optionallysubstituted alkyl, optionally substituted aryl, or optionallysubstituted heteroaryl; R^(g) is optionally substituted alkyl,optionally substituted aryl, or optionally substituted heteroaryl; andR^(h) is hydrogen, —CN, optionally substituted alkyl, optionallysubstituted aryl, or optionally substituted heteroaryl. Substitution mayoccur on any available carbon or nitrogen atom. Exemplary substitutedheterocyclo groups include

and the like. An optionally substituted heterocyclo may be fused to anaryl group to provide an optionally substituted aryl as described above.

The term “alkoxy” as used herein by itself or part of another grouprefers to a haloalkyl, optionally substituted alkyl, optionallysubstituted cycloalkyl, optionally substituted alkenyl or optionallysubstituted alkynyl attached to a terminal oxygen atom. Exemplary alkoxygroups include methoxy, tert-butoxy, —OCH₂CH═CH₂ and the like.

The term “aryloxy” as used herein by itself or part of another grouprefers to an optionally substituted aryl attached to a terminal oxygenatom. Exemplary aryloxy groups include phenoxy and the like.

The term “aralkyloxy” as used herein by itself or part of another grouprefers to an aralkyl attached to a terminal oxygen atom. Exemplaryaralkyloxy groups include benzyloxy and the like.

The term “alkylthio” as used herein by itself or part of another grouprefers to a haloalkyl, aralkyl, optionally substituted alkyl, optionallysubstituted cycloalkyl, optionally substituted alkenyl or optionallysubstituted alkynyl attached to a terminal sulfur atom. Exemplary alkylgroups include —SCH₃ and the like.

The term “halo” or “halogen” as used herein by itself or part of anothergroup refers to fluoro, chloro, bromo or iodo. In one embodiment, thehalo is fluoro or chloro.

The term “amino” as used herein by itself or part of another grouprefers to a radical of formula —NR^(a)R^(b) wherein R^(a) and R^(b) areindependently hydrogen, haloalkyl, aralkyl, optionally substitutedalkyl, optionally substituted cycloalkyl, optionally substitutedheterocyclo, optionally substituted aryl or optionally substitutedheteroaryl; or R^(a) and R^(b) taken together with the nitrogen atom towhich they are attached form a four to seven membered optionallysubstituted heterocyclo. Exemplary amino groups include —NH₂, —N(H)CH₃,—N(CH₃)₂, N(H)CH₂CH₃, N(CH₂CH₃), —N(H)CH₂Ph and the like.

The term “carboxamido” as used herein by itself or part of another grouprefers to a radical of formula —CO-amino. Exemplary carboxamido groupsinclude —CONH₂, —CON(H)CH₃, —CON(H)Ph, —CON(H)CH₂CH₂Ph, —CON(CH₃)₂,CON(H)CHPh₂ and the like.

The term “sulfonamido” as used herein by itself or part of another grouprefers to a radical of formula —SO₂-amino. Exemplary sulfonamido groupsinclude —SO₂NH₂, —SO₂N(H)CH₃, —SO₂N(H)Ph and the like.

The term “about,” as used herein, includes the recited number ±10%.Thus, “about 10” means 9 to 11.

Certain of the compounds of the present disclosure may exist asstereoisomers including optical isomers and conformational isomers (orconformers). The disclosure includes all stereoisomers, both as pureindividual stereoisomer preparations and enriched preparations of each,and both the racemic mixtures of such stereoisomers as well as theindividual diastereomers and enantiomers that may be separated accordingto methods that are well known to those of skill in the art.

Compounds

In certain embodiments, compounds of Formula I are provided:

wherein:

R^(1a), R^(1b), R^(1c), and R^(1d) are independently selected from thegroup consisting of hydrogen, halogen, hydroxy, amino, nitro, cyano,alkoxy, aryloxy, optionally substituted alkyl, haloalkyl, optionallysubstituted cycloalkyl, optionally substituted alkenyl, optionallysubstituted cycloalkenyl, optionally substituted aryl, optionallysubstituted heteroaryl, carboxamido, and sulfonamido;

R² is selected from the group consisting of optionally substituted aryland optionally substituted heteroaryl;

R³ is selected from the group consisting of optionally substitutedalkyl, optionally substituted (cycloalkyl)alkyl, optionally substitutedcycloalkyl, optionally substituted alkenyl, optionally substitutedcycloalkenyl, optionally substituted aryl, and optionally substitutedheteroaryl;

R⁴ is selected from the group consisting of hydrogen and optionallysubstituted alkyl;

R⁵ is selected from the group consisting of:

wherein:

each R^(6a) and R^(6b) is independently selected from the groupconsisting of hydrogen and optionally substituted C₁-C₆ alkyl;

R⁷ is selected from the group consisting of hydrogen, optionallysubstituted C₁-C₆ alkyl, and optionally substituted cycloalkyl;

R^(8a) and R^(8b) are each independently selected from the groupconsisting of hydrogen, optionally substituted C₁-C₆ alkyl, andoptionally substituted cycloalkyl; or

R^(8a) and R^(8b) taken together with the carbon that they are attachedform a 3- to 8-membered optionally substituted cycloalkyl;

W¹ is selected from the group consisting of —OR^(9a) and —NR^(9b)R^(9c);

R^(9a) is hydrogen;

R^(9b) is selected from the group consisting of hydrogen, optionallysubstituted alkyl, optionally substituted cycloalkyl, optionallysubstituted aryl, optionally substituted heteroaryl, —SO₂R^(9d), and—CONR^(9e)R^(9f);

R^(9c) is selected from the group consisting of hydrogen, optionallysubstituted alkyl, optionally substituted cycloalkyl, optionallysubstituted aryl, and optionally substituted heteroaryl; or

R^(9b) and R^(9c) taken together with the nitrogen atom to which theyare attached form a 4- to 8-membered optionally substituted heterocyclo;

R^(9d) is selected from the group consisting of optionally substitutedalkyl and optionally substituted cycloalkyl;

R^(9e) and R^(9f) are each independently selected from the groupconsisting of hydrogen, optionally substituted alkyl, and optionallysubstituted cycloalkyl; or

R^(9e) and R^(9f) taken together with the nitrogen atom to which theyare attached form a 4- to 8-membered optionally substituted heterocyclo;

W² is selected from the group consisting of −OR¹⁰ and —NR^(11a)R^(11b);with the proviso that when W¹ is —OR^(9a) and W² is −OR¹⁰ then at leastone of R⁷, R^(8a), and R^(8b) is other than hydrogen;

R¹⁰ is hydrogen; or

one of R^(9a) and R¹⁰ is hydrogen and the other is a metabolicallycleavable group;

R^(11a) is selected from the group consisting of hydrogen, optionallysubstituted alkyl, optionally substituted cycloalkyl, optionallysubstituted aryl, optionally substituted heteroaryl, —SO₂R^(11c), and—CONR^(11d)R^(11e);

R^(11b) is selected from the group consisting of hydrogen, optionallysubstituted alkyl, optionally substituted cycloalkyl, optionallysubstituted aryl, and optionally substituted heteroaryl; or

R^(11a) and R^(11b) taken together with the nitrogen atom to which theyare attached form a 4- to 8-membered optionally substituted heterocyclo;

R^(11c) is selected from the group consisting of optionally substitutedalkyl and optionally substituted cycloalkyl;

R^(11d) and R^(11e) are each independently selected from the groupconsisting of hydrogen, optionally substituted alkyl, and optionallysubstituted cycloalkyl; or

R^(11d) and R^(11e) together with the nitrogen atom to which they areattached form a 4- to 8-membered optionally substituted heterocyclo;

n is 1, 2, 3, 4, or 5;

each R^(12a), R^(12b), R^(12c) and R^(12d) is independently selectedfrom the group consisting of hydrogen and optionally substituted C₁-C₆alkyl;

R¹³ is selected from the group consisting of hydrogen and optionallysubstituted C₁-C₆ alkyl;

R¹⁴ is selected from the group consisting of hydrogen, optionallysubstituted C₁-C₆ alkyl, and optionally substituted cycloalkyl;

Z is selected from the group consisting of −OR¹⁵ and —NR^(16a)R^(16b);or

Z and R¹⁴ taken together form a carbonyl, i.e., a C═O, group.

R¹⁵ is selected from the group consisting of hydrogen and metabolicallycleavable group;

R^(16a) is selected from the group consisting of —SO₂R^(16c) and—CONR^(16d)R^(16e);

R^(16b) is selected from the group consisting of hydrogen and optionallysubstituted alkyl;

R^(16c) is selected from the group consisting of optionally substitutedalkyl, optionally substituted cycloalkyl, optionally substituted aryl,and optionally substituted heteroaryl;

R^(16d) and R^(16e) are each independently selected from the groupconsisting of hydrogen, optionally substituted alkyl, optionallysubstituted cycloalkyl, optionally substituted aryl, and optionallysubstituted heteroaryl; or

R^(16d) and R^(16e) taken together with the nitrogen atom to which theyare attached form a 4- to 8-membered heterocyclo;

o is 1, 2, or 3;

p is 0, 1, 2, or 3;

each R^(17a), R^(17b), R^(17c) and R^(17d) is independently selectedfrom the group consisting of hydrogen and optionally substituted C₁-C₆alkyl;

R¹⁸ is selected from the group consisting of hydrogen and optionallysubstituted C₁-C₆ alkyl;

R¹⁹ is selected from the group consisting of hydrogen, optionallysubstituted C₁-C₆ alkyl, and optionally substituted cycloalkyl;

R²⁰ is selected from the group consisting of hydrogen, optionallysubstituted C₁-C₆ alkyl, and optionally substituted cycloalkyl;

R^(21a) and R^(21b) are each hydrogen; or

one of R^(21a) and R^(21b) is hydrogen and the other is metabolicallycleavable group;

q is 0, 1, 2, or 3;

r is 1, 2, or 3;

each R^(22a), R^(22b), R^(22c), and R^(22d) is independently selectedfrom the group consisting of hydrogen and optionally substituted C₁-C₆alkyl;

R²³ is selected from the group consisting of hydrogen and optionallysubstituted C₁-C₆ alkyl;

R²⁴ is selected from the group consisting of —SO₂R^(24a) and—CONR^(24b)R^(24c);

R^(24a) is selected from the group consisting of optionally substitutedalkyl, optionally substituted cycloalkyl, optionally substituted aryl,and optionally substituted heteroaryl;

R^(24b) and R^(24a) are each independently selected from the groupconsisting of hydrogen, optionally substituted cycloalkyl, optionallysubstituted aryl, and optionally substituted heteroaryl; or

R^(24b) and R^(24a) taken together with the nitrogen atom to which theyare attached form a 4- to 8-membered heterocyclo;

s and t are each independently 1, 2, or 3;

X is selected from the group consisting of O, S, and NR′;

Y is selected from the group consisting of O, S, and NR″;

R′ is selected from the group consisting of hydrogen, optionallysubstituted alkyl, aralkyl, and optionally substituted cycloalkyl;

R″ is selected from the group consisting of hydrogen, optionallysubstituted alkyl, aralkyl, and optionally substituted cycloalkyl; and

represents a single or a double bond,

or a pharmaceutically acceptable salt, solvate, or prodrug thereof, or acompound having the structure:

or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

In certain embodiments, for the compounds of Formula I,

represents a single or a double bond.

In certain embodiments, the compound of Formula I is a mixture ofstereoisomers, e.g., a mixture of diastereomers and/or enantiomers,e.g., a racemic mixture. In another such embodiment, the compound is amixture of diastereomers. In another such embodiment, the compound is amixture of enantiomers. In particular embodiments, the compound is asingle enantiomer.

In certain embodiments, R⁵ is selected from the group consisting of R5-1and R5-2. In particular embodiments, R⁵ is R5-2 and Z is —OH.

In certain embodiments, compounds of Formula Ia are provided:

wherein R^(1a), R^(1b), R^(1c), R^(1d), R², R³, R⁴, R⁵, X, A and Y havethe meanings as described above for Formula I, or pharmaceuticallyacceptable salt, solvate, or prodrug thereof.

In certain embodiments, compounds of Formula Ib are provided:

wherein R^(1a), R^(1b), R^(1c), R^(1d), R², R³, R⁴, R⁵, X, A and Y havethe meanings as described above for Formula I, or tautomer thereof, or apharmaceutically acceptable salt, solvate, or prodrug thereof.

In certain embodiments, compounds of Formula II-XVII are provided:

wherein R^(1a), R^(1b), R^(1c), R^(1d), R², R³, R⁴, R⁵, X, A and Y havethe meanings as described above for Formula I, or a pharmaceuticallyacceptable salt, solvate, or prodrug thereof.

In some embodiments, compounds of Formula II are provided, whereinR^(1a), R^(1b), R^(1c), R^(1d), R², R³, R⁴, R⁵, X, and Y have themeanings as described above in connection with Formula I, or apharmaceutically acceptable salt, solvate, or prodrug thereof.

In certain embodiments, compounds of Formulae I-XVII or apharmaceutically acceptable salt, solvate, or prodrug thereof areprovided. In some embodiments, compounds of Formula II or apharmaceutically acceptable salt, solvate, or prodrug thereof areprovided, wherein:

a) R^(1a), R^(1b), R^(1c), and R^(1d) are independently selected fromthe group consisting of hydrogen, fluoro, and chloro;

b) R^(1a) and R^(1d) are hydrogen; R^(1b) is selected from the groupconsisting of hydrogen and fluoro; and R^(1c) is selected from the groupconsisting of fluoro and chloro;

c) R² is optionally substituted phenyl;

d) R³ is selected from the group consisting of optionally substitutedalkyl, optionally substituted (cycloalkyl)alkyl, and optionallysubstituted cycloalkyl;

e) R⁴ is hydrogen;

f) X is NH;

g) X is O;

h) X is S;

i) Y is O;

j) Y is S;

k) Y is NH; or

l) X and Y are NH;

or any combination thereof.

In certain embodiments, compounds of Formula II or a pharmaceuticallyacceptable salt, solvate, or prodrug thereof are provided, wherein R⁵ isR5-1; R^(6a) and R^(6b) are hydrogen; R⁷ is C₁-C₄ alkyl; R^(8a) andR^(8b) are hydrogen; W is —OR¹⁰, R⁹ and R¹⁰ are hydrogen; and n is 2.

In certain embodiments, compounds of Formula II or a pharmaceuticallyacceptable salt, solvate, or prodrug thereof are provided, wherein R⁵ isR5-1; R^(6a) and R^(6b) are hydrogen; R⁷ is C₁-C₄ alkyl; R^(8a) andR^(8b) are hydrogen; W is —NR^(11a)R^(11b), R⁹ is hydrogen; and n is 2.

In certain embodiments, the compounds of Formula II or apharmaceutically acceptable salt, solvate, or prodrug thereof areprovided, wherein R⁵ is R5-1; R^(6a) and R^(6b) are hydrogen; R⁷ isC₁-C₄ alkyl; R^(8a) and R^(8b) are hydrogen; W is −OR¹⁰, one of R⁹ andR¹⁰ is hydrogen and the other is a metabolically cleavable group; and nis 2.

In certain embodiments, compounds of Formula II or a pharmaceuticallyacceptable salt, solvate, or prodrug thereof are provided, wherein R⁵ isR5-2; R^(12a), R^(12b), R^(12c), and R^(12d) are each hydrogen; R¹³ ishydrogen; Z is —OR¹⁵ and R¹⁵ is hydrogen; o is 1 or 2; and p is 1 or 2.

In certain embodiments, compounds of Formula II or a pharmaceuticallyacceptable salt, solvate, or prodrug thereof are provided, wherein R⁵ isR5-2; R^(12a), R^(12b), R^(12c), and R^(12d) are each hydrogen; R¹³ ishydrogen; Z is —NR^(16a)R^(16b); o is 1 or 2; and p is 1 or 2.

In certain embodiments, compounds of Formula II or a pharmaceuticallyacceptable salt, solvate, or prodrug thereof are provided, wherein R⁵ isR5-2; R^(12a), R^(12b), R^(12c), and R^(12d) are each hydrogen; R¹³ ishydrogen; Z is —OR¹⁵ and R¹⁵ a metabolically cleavable group; o is 1 or2; and p is 1 or 2.

In certain embodiments, compounds of Formula II or a pharmaceuticallyacceptable salt, solvate, or prodrug thereof are provided, wherein R⁵ isR5-3; R^(17a), R^(17b), R^(17c), and R^(17d) are each hydrogen; R¹⁸,R¹⁹, and R²⁰ are hydrogen; R^(21a) and R^(21b) are hydrogen; and q and rare 1.

In certain embodiments, compounds of Formula II or a pharmaceuticallyacceptable salt, solvate, or prodrug thereof are provided, wherein R⁵ isR5-3; R^(17a), R^(17b), R^(17c), and R^(17d) are each hydrogen; R¹⁸,R¹⁹, and R²⁰ are hydrogen; one of R^(21a) and R^(21b) is hydrogen andthe other is a metabolically cleavable group; and q and r are 1.

In certain embodiments, compounds of Formula II or a pharmaceuticallyacceptable salt, solvate, or prodrug thereof are provided, wherein R² isan optionally substituted aryl having the Formula R2-1:

and R^(25a), R^(25b), R^(25c), R^(25d), and R^(25e) are eachindependently selected from the group consisting of hydrogen, halogen,hydroxy, nitro, amino, cyano, alkoxy, optionally substituted alkyl,haloalkyl, optionally substituted aryl, and optionally substitutedheteroaryl. In particular embodiments, R^(25a) is selected from thegroup consisting of hydrogen and fluoro; R^(25b) is chloro; R^(25c) isselected from the group consisting of hydrogen and fluoro; and R^(25d)and R^(25e) are hydrogen.

In certain embodiments, compounds of Formula II or a pharmaceuticallyacceptable salt, solvate, or prodrug thereof are provided, wherein R⁵ isselected from the group consisting of:

including stereoisomers, e.g., enantiomers, thereof, wherein:

R⁷ is optionally substituted C₁-C₄ alkyl;

R^(9a) and R¹⁰ are each hydrogen; or

one of R^(9a) and R¹⁰ is hydrogen and the other is a metabolicallycleavable group;

R^(9b) is selected from the group consisting of hydrogen, optionallysubstituted alkyl, optionally substituted cycloalkyl, optionallysubstituted aryl, optionally substituted heteroaryl, —SO₂R^(9d), and—CONR^(9e)R^(9f);

R^(9c) is selected from the group consisting of hydrogen, optionallysubstituted alkyl, optionally substituted cycloalkyl, optionallysubstituted aryl, and optionally substituted heteroaryl; or

R^(9b) and R^(9c) taken together with the nitrogen atom to which theyare attached form a 4- to 8-membered optionally substituted heterocyclo;

R^(9d) is selected from the group consisting of optionally substitutedalkyl and optionally substituted cycloalkyl;

R^(9e) and R^(9f) are each independently selected from the groupconsisting of hydrogen, optionally substituted alkyl, and optionallysubstituted cycloalkyl; or

R^(9e) and R^(9f) taken together with the nitrogen atom to which theyare attached form a 4- to 8-membered optionally substituted heterocyclo;

R^(11a) is selected from the group consisting of hydrogen, optionallysubstituted alkyl, optionally substituted cycloalkyl, optionallysubstituted aryl, optionally substituted heteroaryl, —SO₂R^(1lc), and—CONR^(11d)R^(11e);

R^(11b) is selected from the group consisting of hydrogen, optionallysubstituted alkyl, optionally substituted cycloalkyl, optionallysubstituted aryl, and optionally substituted heteroaryl; or

R^(11a) and R^(11b) taken together with the nitrogen atom to which theyare attached form a 4- to 8-membered optionally substituted heterocyclo;

R^(11c) is selected from the group consisting of optionally substitutedalkyl and optionally substituted cycloalkyl;

-   -   R^(11d) and R^(11e) are each independently selected from the        group consisting of hydrogen, optionally substituted alkyl, and        optionally substituted cycloalkyl; or

R^(11d) and R^(11e) taken together with the nitrogen atom to which theyare attached form a 4- to 8-membered optionally substituted heterocyclo;

R¹⁴ is selected from the group consisting of hydrogen, C₁-C₄ alkyl, orC₃-C₆ cycloalkyl;

R¹⁵ is hydrogen or a metabolically cleavable group;

R^(16a) is selected from the group consisting of —SO₂R^(16c) and—CONR^(16d)R^(16e);

R^(16b) is selected from the group consisting of hydrogen and optionallysubstituted alkyl;

R^(16c) is selected from the group consisting of optionally substitutedalkyl, optionally substituted cycloalkyl, optionally substituted aryl,and optionally substituted heteroaryl;

R^(16d) and R^(16e) are each independently selected from the groupconsisting of hydrogen, optionally substituted alkyl, optionallysubstituted cycloalkyl, optionally substituted aryl, and optionallysubstituted heteroaryl; or

R^(16d) and R^(16e) taken together with the nitrogen atom to which theyare attached form a 4- to 8-membered heterocyclo;

R¹⁹ is selected from the group consisting of hydrogen, optionallysubstituted C₁-C₆ alkyl, and optionally substituted cycloalkyl;

R²⁰ is selected from the group consisting of hydrogen, optionallysubstituted C₁-C₆ alkyl, and optionally substituted cycloalkyl;

R^(21a) and R^(21b) are each hydrogen; or

one of R^(21a) and R^(21b) is hydrogen and the other is metabolicallycleavable group;

R²⁴ is selected from the group consisting of —SO₂R^(24a) and—CONR^(24b)R^(24c);

R^(24a) is selected from the group consisting of optionally substitutedalkyl, optionally substituted cycloalkyl, optionally substituted aryl,and optionally substituted heteroaryl; and

R^(24b) and R^(24c) are each independently selected from the groupconsisting of hydrogen, optionally substituted cycloalkyl, optionallysubstituted aryl, and optionally substituted heteroaryl, or

R^(24b) and R^(24c) taken together with the nitrogen atom to which theyare attached form a 4- to 8-membered heterocyclo.

In certain embodiments, R⁵ is selected from the group consisting ofR5-5, R5-6, R5-10, R5-11, R5-12, R5-13, and R5-14.

In certain embodiments, R⁵ is selected from the group consisting ofR5-10 and R5-12 and R¹⁴ is hydrogen or methyl and R¹⁵ is hydrogen.

In certain embodiments, compounds of Formula II or a pharmaceuticallyacceptable salt, solvate, or prodrug thereof are provided, wherein R⁵ isselected from the group consisting of:

wherein:

R⁷ is selected from the group consisting of methyl, ethyl, propyl,isopropyl, and cyclopropyl; and

R^(8a) and R^(8b) are each independently selected from the groupconsisting of hydrogen, methyl, ethyl, propyl, isopropyl, andcyclopropyl.

In certain embodiments, compounds of Formula II or a pharmaceuticallyacceptable salt, solvate, or prodrug thereof are provided, wherein R⁵ isselected from the group consisting of:

wherein:

R⁷ is selected from the group consisting of methyl, ethyl, propyl,isopropyl, and cyclopropyl;

R^(8a) and R^(8b) are each independently is selected from the groupconsisting of hydrogen, methyl, ethyl, propyl, isopropyl, andcyclopropyl;

R^(9d) is selected from the group consisting of methyl, trifluoromethyl,ethyl, propyl, isopropyl, and cyclopropyl; and

R^(11c) is selected from the group consisting of methyl,trifluoromethyl, ethyl, propyl, isopropyl, and cyclopropyl.

In certain embodiments, compounds of Formula II or a pharmaceuticallyacceptable salt, solvate, or prodrug thereof are provided, wherein R⁵ isselected from the group consisting of:

wherein:

R⁷ is selected from the group consisting of methyl, ethyl, propyl,isopropyl, and cyclopropyl;

R^(8a) and R^(8b) are each independently is selected from the groupconsisting of hydrogen, methyl, ethyl, propyl, isopropyl, andcyclopropyl;

R^(9e) is selected from the group consisting of methyl, trifluoromethyl,ethyl, propyl, isopropyl, and cyclopropyl; and

R^(11d) is selected from the group consisting of methyl,trifluoromethyl, ethyl, propyl, isopropyl, and cyclopropyl.

In certain embodiments, compounds of Formula II or a pharmaceuticallyacceptable salt, solvate, or prodrug thereof are provided, wherein R⁵ isselected from the group consisting of:

wherein:

R¹⁴ is selected from the group consisting of methyl, ethyl, propyl,isopropyl, and cyclopropyl; and

R¹⁹ and R²⁰ are each independently is selected from the group consistingof hydrogen, methyl, ethyl, propyl, isopropyl, and cyclopropyl.

In certain embodiments, compounds of Formula II or a pharmaceuticallyacceptable salt, solvate, or prodrug thereof are provided, wherein R⁵ isselected from the group consisting of:

wherein:

R¹⁴ is selected from the group consisting of methyl, ethyl, propyl,isopropyl, and cyclopropyl; and

R^(16c) is selected from the group consisting of methyl,trifluoromethyl, ethyl, propyl, isopropyl, and cyclopropyl.

In certain embodiments, compounds of Formula II or a pharmaceuticallyacceptable salt, solvate, or prodrug thereof are provided, wherein R⁵ isselected from the group consisting of:

wherein:

R¹⁴ is selected from the group consisting of methyl, ethyl, propyl,isopropyl, and cyclopropyl; and

R^(16d) is selected from the group consisting of methyl,trifluoromethyl, ethyl, propyl, isopropyl, and cyclopropyl.

In another embodiment, compounds of Formula XVIIIa are provided:

wherein:

R^(1b) and R^(1c) are independently selected from the group consistingof hydrogen, fluoro, and chloro;

R³ is selected from the group consisting of optionally substitutedalkyl, optionally substituted (cycloalkyl)alkyl, and optionallysubstituted cycloalkyl;

R^(26a), R^(26b), and R^(26c) are independently selected from the groupconsisting of hydrogen, fluoro, and chloro; and

R²⁷ is selected from the group consisting of:

wherein:

R⁷ is optionally substituted C₁-C₄ alkyl;

W² is selected from the group consisting of —OR¹⁰ and —NR^(11a)R^(11b);

R^(9a) and R¹⁰ are each hydrogen; or

one of R^(9a) and R¹⁰ is hydrogen and the other is a metabolicallycleavable group;

R^(11a) is selected from the group consisting of hydrogen, optionallysubstituted alkyl, optionally substituted cycloalkyl, optionallysubstituted aryl, optionally substituted heteroaryl, —SO₂R^(11c), and—CONR^(11d)R^(11e);

R^(11b) is selected from the group consisting of hydrogen, optionallysubstituted alkyl, optionally substituted cycloalkyl, optionallysubstituted aryl, and optionally substituted heteroaryl; or

R^(11a) and R^(11b) taken together with the nitrogen atom to which theyare attached form a 4- to 8-membered optionally substituted heterocyclo;

R^(11c) is selected from the group consisting of optionally substitutedalkyl and optionally substituted cycloalkyl;

R^(11d) and R^(11e) are each independently selected from the groupconsisting of hydrogen, optionally substituted alkyl, and optionallysubstituted cycloalkyl; or

R^(11d) and R^(11e) taken together with the nitrogen atom to which theyare attached form a 4- to 8-membered optionally substituted heterocyclo;

R¹⁴ is selected from the group consisting of hydrogen, optionallysubstituted C₁-C₄ alkyl, and optionally substituted cycloalkyl;

Z is selected from the group consisting of —OR¹⁵ and —NR^(16a)R^(16b);

R¹⁵ is selected from the group consisting of hydrogen and metabolicallycleavable group;

R^(16a) is selected from the group consisting of —SO₂R^(16c) and—CONR^(16d)R^(16e);

R^(16b) is selected from the group consisting of hydrogen and optionallysubstituted alkyl;

R^(16c) is selected from the group consisting of optionally substitutedalkyl, optionally substituted cycloalkyl, optionally substituted aryl,and optionally substituted heteroaryl;

R^(16d) and R^(16e) are each independently selected from the groupconsisting of hydrogen, optionally substituted alkyl, optionallysubstituted cycloalkyl, optionally substituted aryl, and optionallysubstituted heteroaryl; or

R^(16d) and R^(16e) taken together with the nitrogen atom to which theyare attached form a 4- to 8-membered heterocyclo;

R¹⁹ is selected from the group consisting of hydrogen, optionallysubstituted C₁-C₆ alkyl, and optionally substituted cycloalkyl;

R²⁰ is selected from the group consisting of hydrogen, optionallysubstituted C₁-C₆ alkyl, and optionally substituted cycloalkyl;

R^(21a) and R^(21b) are each hydrogen; or

one of R^(21a) and R^(21b) is hydrogen and the other is metabolicallycleavable group;

or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

In another embodiment, compounds of Formula XVIIIb are provided:

wherein R^(1b), R^(1b), R³, R^(26a), R^(26b), R^(26c), and R²⁷ have themeanings as described above for Formula XVIIIa, or a pharmaceuticallyacceptable salt, solvate, or prodrug thereof.

In another embodiment, compounds of Formula XVIIIc are provided:

wherein R^(1b), R^(1b), R³, R^(26a), R^(26b), R^(26c), and R²⁷ have themeanings as described above for Formula XVIIIa, or a pharmaceuticallyacceptable salt, solvate, or prodrug thereof.

In certain embodiments, R²⁷ is selected from the group consisting ofR27-2, R27-3, R27-5, R27-6, R27-8, R27-9, R27-11, R27-12, R27-14,R27-15, R27-16, R27-17, R27-19, R27-20, R27-21, R27-22, R27-24, R27-25,R27-27, R27-29, R27-30, R27-31, and R27-32. In certain embodiments, R²⁷is selected from the group consisting of R27-2, R27-3, R27-5, and R27-6,R27-8, R27-9, R27-14, R27-15, R27-16, and R27-17. In certainembodiments, R²⁷ is a hydroxycycloalkyl group.

In certain embodiments, R^(9a) is hydrogen; W² is OH; Z is OH; R⁷ isC₁-C₄ alkyl, e.g., methyl, ethyl, propyl, or isopropyl, or cyclopropyl;R¹⁴, R¹⁹, and R²⁰ are each independently hydrogen, C₁-C₄ alkyl, e.g.,methyl, ethyl, propyl, or isopropyl, or cyclopropyl; and R^(21a) andR^(21b) are each hydrogen.

In certain embodiments, R^(9a) is hydrogen, R⁷ is hydrogen, C₁-C₄ alkyl,or cyclopropyl; W² is —NHR^(11a); R^(11a) is C₁-C₄ alkyl, e.g., methyl,trifluoromethyl, ethyl, propyl, or isopropyl, or cyclopropyl; R¹⁴ ishydrogen, C₁-C₄ alkyl, e.g., methyl, ethyl, propyl, or isopropyl, orcyclopropyl; Z is —NHSO₂R^(16c) or —NHCONHR^(16d); and R^(16c) andR^(16d) are each independently optionally substituted C₁-C₄ alkyl, e.g.,methyl, trifluoromethyl, ethyl, propyl, or isopropyl, or cyclopropyl.

In certain embodiments, compounds of Formulae XIX-XXXIV are provided:

wherein R^(1b), R^(1c), R³, R^(26a), R^(26b), R^(26c), and R²⁷ have themeanings as described above in connection with Formula XVIIIa, or apharmaceutically acceptable salt, solvate, or prodrug thereof.

In certain embodiments, compounds of Formula XIX are provided, whereinR^(1b), R^(1c), R³, R^(26a), R^(26b), R^(26c), and R²⁷ have the meaningsas described above in connection with Formula XVIIIa, or apharmaceutically acceptable salt, solvate, or prodrug thereof.

In certain embodiments, compounds of Formula XIX are provided, whereinR²⁷ is selected from the group consisting of:

wherein:

R⁷ is C₁-C₄ alkyl;

R^(9a) and R¹⁰ are hydrogen; or

one of R^(9a) and R¹⁰ is hydrogen and the other is metabolicallycleavable group;

R^(11a) and R^(11b) are each independently selected from the groupconsisting of hydrogen, optionally substituted C₁-C₄ alkyl, optionallysubstituted cycloalkyl, optionally substituted aryl, and optionallysubstituted heteroaryl; or

R^(11a) and R^(11b) taken together with the nitrogen atom to which theyare attached form a 4- to 8-membered optionally substituted heterocyclo;

R¹⁴ is selected from the group consisting of hydrogen and C₁-C₄ alkyl;

R¹⁵ is hydrogen or a metabolically cleavable group; and

R^(16a) is selected from the group consisting of —SO₂R^(16c) and—CONR^(16d)R^(16e);

R^(16c) is selected from the group consisting of optionally substitutedC₁-C₄ alkyl or cyclopropyl;

R^(16d) and R^(16e) are each independently selected from the groupconsisting of hydrogen, optionally substituted C₁-C₄ alkyl orcyclopropyl; or

R^(16d) and R^(16e) taken together with the nitrogen atom to which theyare attached form a 4- to 8-membered heterocyclo,

or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

In certain embodiments, compounds of Formula XIX are provided, whereinR²⁷ is selected from the group consisting of:

wherein:

R⁷ is optionally substituted C₁-C₄ alkyl;

W² is selected from the group consisting of —OR¹⁰ and —NR^(11a)R^(11b);

R^(9a) and R¹⁰ are each hydrogen; or

one of R^(9a) and R¹⁰ is hydrogen and the other is a metabolicallycleavable group;

R^(11a) is selected from the group consisting of hydrogen, optionallysubstituted alkyl, optionally substituted cycloalkyl, optionallysubstituted aryl, optionally substituted hetero aryl, —SO₂R^(11c), and—CONR^(11d)R^(11e);

R^(11b) is selected from the group consisting of hydrogen, optionallysubstituted alkyl, optionally substituted cycloalkyl, optionallysubstituted aryl, and optionally substituted heteroaryl; or

R^(11c) and R^(11b) taken together with the nitrogen atom to which theyare attached form a 4- to 8-membered optionally substituted heterocyclo;

R^(11c) is selected from the group consisting of optionally substitutedalkyl and optionally substituted cycloalkyl;

R^(11d) and R^(11e) are each independently selected from the groupconsisting of hydrogen, optionally substituted alkyl, and optionallysubstituted cycloalkyl; or

R^(11d) and R^(11e) taken together with the nitrogen atom to which theyare attached form a 4- to 8-membered optionally substituted heterocyclo;

or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

In certain embodiments, compounds of Formula XIX are provided, whereinR²⁷ is selected from the group consisting of:

In certain embodiments, compounds of Formula XIX are provided, whereinR²⁷ is selected from the group consisting of:

wherein R^(11a) and R^(11b) taken together with the nitrogen to whichthey are attached form a 5- or 6-membered optionally substitutedheterocyclo.

In certain embodiments, compounds of Formula XIX are provided, whereinR²⁷ is selected from the group consisting of:

wherein:R¹⁴ is selected from the group consisting of hydrogen and C₁-C₄ alkyl;andR¹⁵ is a metabolically cleavable group.

In certain embodiments, compounds of Formulae II and XIX are provided,wherein R¹⁵ is a metabolically cleavable group selected from the groupconsisting of:

wherein:

each R^(28a) and R^(28b) is independently selected from the groupconsisting of hydrogen, optionally substituted alkyl, and aralkyl;

R^(29a) and R^(29b) are each selected from the group consisting ofhydrogen and optionally substituted alkyl;

v is 1, 2, 3, or 4; and

R^(30a) and R^(30b) are each selected from the group consisting ofhydrogen, optionally substituted alkyl, aralkyl, optionally substitutedaryl, and monovalent pharmaceutically acceptable cation; or

taken together R^(30a) and R^(30b) represent a divalent pharmaceuticallyacceptable cation or an optionally substituted alkylenyl.

In certain embodiments, R¹⁵ is the residue of a natural or unnaturalamino acid. In other embodiments, R¹⁵ is the residue of glycine,isoleucine alanine, leucine, asparagine, lysine, aspartic acid,methionine, cysteine, phenylalanine, glutamic acid, threonine,glutamine, tryptophan, valine, proline, serine, tyrosine, arginine, andhistidine

In certain embodiments, compounds of Formulae II and XIX are provided,wherein R³ is C₁-C₁₀ alkyl.

In certain embodiments, compounds of Formulae II and XIX are provided,wherein R³ is selected from the group consisting of —CH₂C(CH₃)₃,—CH₂C(CH₃)₂CH₂CH₃, —CH₂C(CH₃)₂CH₂CH₂CH₃, —CH₂C(CH₃)₂CH₂CH₂CH₂CH₃,—CH₂C(CH₂CH₃)₂CH₃, and —CH₂C(CH₃)₂CH₂CH(CH₃)₂. In certain embodiments,R³ is —CH₂C(CH₃)₃

In certain embodiments, compounds of Formula I are provided having thestructure:

or a pharmaceutically acceptable salt, solvate, or prodrug thereof(“Ms”=—SO₂CH₃).

In certain embodiments, compounds of Formula I are provided having thefollowing structures:

or a pharmaceutically acceptable salt, solvate, or prodrug thereof(“Ms”=—SO₂CH₃).

In certain embodiments, compounds of Formula I are provided having thefollowing structures, which contain metabolically cleavable amino acidesters or phosphate esters as prodrugs:

or a pharmaceutically acceptable salt or solvate thereof.

In certain embodiments, compounds having the following structure:

or a pharmaceutically acceptable salt or solvate thereof are provided.

In certain embodiments, compounds having the following structure:

or a pharmaceutically acceptable salt or solvate thereof are provided.

In certain embodiments, compounds having the following structure:

or a pharmaceutically acceptable salt or solvate thereof are provided.

In certain embodiments, compounds having the following structure:

or a pharmaceutically acceptable salt or solvate thereof are provided.

In certain embodiments, compounds having the following structure:

or a pharmaceutically acceptable salt or solvate thereof are provided.

In certain embodiments, compounds having the following structure:

or a pharmaceutically acceptable salt or solvate thereof are provided.

In certain embodiments, compounds having the following structure:

or a pharmaceutically acceptable salt or solvate thereof are provided.

In certain embodiments, compounds having the following structure:

or a pharmaceutically acceptable salt or solvate thereof are provided.

The compounds and processes provided herein will be better understood inconnection with the following synthetic schemes which illustrate themethods by which the compounds provided herein may be prepared. Startingmaterials can be obtained from commercial sources or prepared bywell-established literature methods known to those of ordinary skill inthe art. It will be readily apparent to one of ordinary skill in the artthat the compounds defined above can be synthesized by substitution ofthe appropriate reagents and agents in the syntheses shown below.

Compounds of Formula Ia wherein Y is NH can be synthesized as describedin Schemes 2 and 3.

Compounds of Formula Ia can be separated by chiral resolution methodswell know in the art, e.g., chiral column chromatography, to givecompounds of Formulae II-XVII. Suitable chiral columns for use in chiralresolutions include, for example, Daicel CHIRALCEL® OD-H, DaicelCHIRAKPAK® AD-H and Regis Technologies ULMO chiral columns. Other chiralresolution methods are also possible. Compounds of Formulae II-XVII canalso be prepared by asymmetric synthetic methods. For example, compoundsof Formula II, wherein Y is NH, can be synthesized by using a asymmetric1,3-dipolar cycloaddition as the key step as previously described (SeeU.S. Pat. Nos. 7,759,383 B2 and 7,737,174 B2, and Ding et al., J. Am.Chem. Soc. 127:10130-10131 (2005)) (Scheme 4).

Briefly, compound A reacts with aldehyde B to give C. Compound C reactswith aldehyde E and compound D to give F (a compound of Formula Iwherein R″ is aralkyl). Treatment of F with Pb(OAc)₄ or CAN gives thecompound of Formula II wherein Y is NH.

Methods

In some embodiments, compounds provided herein induce cell cycle arrestand/or apoptosis and also potentiate the induction of cell cycle arrestand/or apoptosis either alone or in response to additional apoptosisinduction signals. Therefore, it is contemplated that these compoundssensitize cells to induction of cell cycle arrest and/or apoptosis,including cells that are resistant to such inducing stimuli. Byinhibiting the interaction between p53 or p53-related proteins and MDM2or MDM2-related proteins, the compounds provided herein can be used toinduce apoptosis in any disorder that can be treated, ameliorated, orprevented by the induction of apoptosis. In one embodiment, theinhibitors can be used to induce apoptosis in cells comprisingfunctional p53 or p53-related proteins.

In another embodiment, the disclosure pertains to modulating apoptosiswith compounds provided herein in combination with one or moreadditional apoptosis-modulating agents. Examples of apoptosis-modulatingagents include, but are not limited to, Fas/CD95, TRAMP, TNF RI, DR1,DR2, DR3, DR4, DRS, DR6, FADD, RIP, TNFα, Fas ligand, TRAIL, antibodiesto TRAIL-R1 or TRAIL-R2, Bcl-2, p53, BAX, BAD, Akt, CAD, PI3 kinase,PP1, and caspase proteins. Other agents involved in the initiation,decision and degradation phase of apoptosis are also included. Examplesof apoptosis-modulating agents include agents, the activity, presence,or change in concentration of which, can modulate apoptosis in asubject. Apoptosis-modulating agents include those which are inducers ofapoptosis, such as TNF or a TNF-related ligand, particularly a TRAMPligand, a Fas/CD95 ligand, a TNFR-1 ligand, or TRAIL.

In some embodiments, the compositions and methods provided herein areused to treat diseased cells, tissues, organs, or pathologicalconditions and/or disease states in an animal (e.g., a mammalian patientincluding, but not limited to, humans and veterinary animals). In thisregard, various diseases and pathologies are amenable to treatment orprophylaxis using the present methods and compositions. A non-limitingexemplary list of these diseases and conditions includes, but is notlimited to, breast cancer, prostate cancer, lymphoma, skin cancer,pancreatic cancer, colon cancer, melanoma, malignant melanoma, ovariancancer, brain cancer, primary brain carcinoma, head-neck cancer, glioma,glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer,head or neck carcinoma, breast carcinoma, ovarian carcinoma, lungcarcinoma, small-cell lung carcinoma, Wilms' tumor, cervical carcinoma,testicular carcinoma, bladder carcinoma, pancreatic carcinoma, stomachcarcinoma, colon carcinoma, prostatic carcinoma, genitourinarycarcinoma, thyroid carcinoma, esophageal carcinoma, myeloma, multiplemyeloma, adrenal carcinoma, renal cell carcinoma, endometrial carcinoma,adrenal cortex carcinoma, malignant pancreatic insulinoma, malignantcarcinoid carcinoma, choriocarcinoma, mycosis fungoides, malignanthypercalcemia, cervical hyperplasia, leukemia, acute lymphocyticleukemia, chronic lymphocytic leukemia (CLL) including B-CLL, acutemyelogenous leukemia, chronic myelogenous leukemia, chronic granulocyticleukemia, acute granulocytic leukemia, hairy cell leukemia,neuroblastoma, sarcoma such as liposarcoma malignant fibroushistiocytoma, osteosarcoma, Ewing's sarcoma, leiomyosarcoma, andrhabdomyosarcoma, Kaposi's sarcoma, polycythemia vera, essentialthrombocytosis, Hodgkin's disease, non-Hodgkin's lymphoma, soft-tissuesarcomas such as lipoma, and malignant Schwannoma, osteogenic sarcoma,primary macroglobulinemia, and retinoblastoma, and the like, T and Bcell mediated autoimmune diseases; inflammatory diseases; infections;hyperproliferative diseases; AIDS; degenerative conditions, vasculardiseases, and the like. In some embodiments, the cancer cells beingtreated are metastatic. In other embodiments, the cancer cells beingtreated are resistant to other anticancer agents.

In some embodiments, the compositions and methods provided herein areused to treat cancers that express functional or wild type p53 orp53-related proteins. In some embodiments, the compositions and methodsprovided herein are used to treat cancers that express elevated levelsof MDM2 or MDM2-related proteins.

In some embodiments, the methods, compounds, and compositions providedherein can be used to treat a patient having a sarcoma, including, forexample, liposarcoma, malignant fibrous histiocytoma, osteosarcoma, andrhabdomyosarcoma. In some embodiments, the methods, compounds, andcompositions provided herein can be used to treat a patient having asoft tissue tumor, including, for example, Ewing's sarcoma,leiomyosarcoma, lipoma, and malignant Schwannomas. In some embodiments,the methods, compounds, and compositions provided herein can be used totreat a patient having lung, breast, liver, or colon cancer. In someembodiments, the methods, compounds, and compositions provided hereincan be used to treat a patient having B-cell chronic lymphocyticleukemia and acute myeloid leukemia.

In some embodiments, infections suitable for treatment with thecompositions and methods provided herein include, but are not limitedto, infections caused by viruses, bacteria, fungi, mycoplasma, prions,and the like.

In some embodiments, methods are provided for administering an effectiveamount of a compound provided herein and at least one additionaltherapeutic agent (including, but not limited to, chemotherapeuticantineoplastics, apoptosis-modulating agents, antimicrobials,antivirals, antifungals, and anti-inflammatory agents) and/ortherapeutic technique (e.g., surgical intervention, and/orradiotherapies). In a particular embodiment, the additional therapeuticagent(s) is a anticancer agent.

A number of suitable therapeutic or anticancer agents are contemplatedfor use in the methods provided herein. Indeed, the methods providedherein can include but are not limited to, administration of numeroustherapeutic agents such as: agents that induce apoptosis;polynucleotides (e.g., anti-sense, ribozymes, siRNA); polypeptides(e.g., enzymes and antibodies); biological mimetics (e.g., gossypol orBH3 mimetics); agents that bind (e.g., oligomerize or complex) with aBcl-2 family protein such as Bax; alkaloids; alkylating agents;antitumor antibiotics; antimetabolites; hormones; platinum compounds;monoclonal or polyclonal antibodies (e.g., antibodies conjugated withanticancer drugs, toxins, defensins), toxins; radionuclides; biologicalresponse modifiers (e.g., interferons (e.g., IFN-α) and interleukins(e.g., IL-2)); adoptive immunotherapy agents; hematopoietic growthfactors; agents that induce tumor cell differentiation (e.g.,all-trans-retinoic acid); gene therapy reagents (e.g., antisense therapyreagents and nucleotides); tumor vaccines; angiogenesis inhibitors;proteosome inhibitors: NF-κB modulators; anti-CDK compounds; HDACinhibitors; and the like. Numerous other examples of therapeutic agentssuch as chemotherapeutic compounds and anticancer therapies suitable forco-administration with the disclosed compounds are known to thoseskilled in the art.

In certain embodiments, anticancer agents comprise agents that induce orstimulate apoptosis. Agents that induce or stimulate apoptosis include,for example, agents that interact with or modify DNA, such as byintercalating, cross-linking, alkylating, or otherwise damaging orchemically modifying DNA. Agents that induce apoptosis include, but arenot limited to, radiation (e.g., X-rays, gamma rays, UV); tumor necrosisfactor (TNF)-related factors (e.g., TNF family receptor proteins, TNFfamily ligands, TRAIL, antibodies to TRAIL-R1 or TRAIL-R2); kinaseinhibitors (e.g., epidermal growth factor receptor (EGFR) kinaseinhibitor. Additional anticancer agents include: vascular growth factorreceptor (VGFR) kinase inhibitor, fibroblast growth factor receptor(FGFR) kinase inhibitor, platelet-derived growth factor receptor (PDGFR)kinase inhibitor, and Bcr-Abl kinase inhibitors (such as GLEEVEC));antisense molecules; antibodies (e.g., HERCEPTIN, RITUXAN, ZEVALIN, andAVASTIN); anti-estrogens (e.g., raloxifene and tamoxifen);anti-androgens (e.g., flutamide, bicalutamide, finasteride,aminoglutethamide, ketoconazole, and corticosteroids); cyclooxygenase 2(COX-2) inhibitors (e.g., celecoxib, meloxicam, NS-398, andnon-steroidal anti-inflammatory drugs (NSAIDs)); anti-inflammatory drugs(e.g., butazolidin, DECADRON, DELTASONE, dexamethasone, dexamethasoneintensol, DEXONE, HEXADROL, hydroxychloroquine, METICORTEN, ORADEXON,ORASONE, oxyphenbutazone, PEDIAPRED, phenylbutazone, PLAQUENIL,prednisolone, prednisone, PRELONE, and TANDEARIL); and cancerchemotherapeutic drugs (e.g., irinotecan (CAMPTOSAR), CPT-11,fludarabine (FLUDARA), dacarbazine (DTIC), dexamethasone, mitoxantrone,MYLOTARG, VP-16, cisplatin, carboplatin, oxaliplatin, 5-FU, doxorubicin,gemcitabine, bortezomib, gefitinib, bevacizumab, TAXOTERE or TAXOL);cellular signaling molecules; ceramides and cytokines; staurosporine,and the like.

In still other embodiments, the compositions and methods provided hereininclude one or more compounds provided herein and at least oneanti-hyperproliferative or antineoplastic agent selected from alkylatingagents, antimetabolites, and natural products (e.g., herbs and otherplant and/or animal derived compounds).

Alkylating agents suitable for use in the present compositions andmethods include, but are not limited to: 1) nitrogen mustards (e.g.,mechlorethamine, cyclophosphamide, ifosfamide, melphalan (L-sarcolysin);and chlorambucil); 2) ethylenimines and methylmelamines (e.g.,hexamethylmelamine and thiotepa); 3) alkyl sulfonates (e.g., busulfan);4) nitrosoureas (e.g., carmustine (BCNU); lomustine (CCNU); semustine(methyl-CCNU); and streptozocin (streptozotocin)); and 5) triazenes(e.g., dacarbazine (DTIC; dimethyltriazenoimid-azolecarboxamide).

In some embodiments, antimetabolites suitable for use in the presentcompositions and methods include, but are not limited to: 1) folic acidanalogs (e.g., methotrexate (amethopterin)); 2) pyrimidine analogs(e.g., fluorouracil (5-fluorouracil; 5-FU), floxuridine(fluorode-oxyuridine; FudR), and cytarabine (cytosine arabinoside)); and3) purine analogs (e.g., mercaptopurine (6-mercaptopurine; 6-MP),thioguanine (6-thioguanine; TG), and pentostatin (2′-deoxycoformycin)).

In still further embodiments, chemotherapeutic agents suitable for usein the compositions and methods of the present disclosure include, butare not limited to: 1) vinca alkaloids (e.g., vinblastine (VLB),vincristine); 2) epipodophyllotoxins (e.g., etoposide and teniposide);3) antibiotics (e.g., dactinomycin (actinomycin D), daunorubicin(daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin(mithramycin), and mitomycin (mitomycin C)); 4) enzymes (e.g.,L-asparaginase); 5) biological response modifiers (e.g.,interferon-alfa); 6) platinum coordinating complexes (e.g., cisplatin(cis-DDP) and carboplatin); 7) anthracenediones (e.g., mitoxantrone); 8)substituted ureas (e.g., hydroxyurea); 9) methylhydrazine derivatives(e.g., procarbazine (N-methylhydrazine; MIH)); 10) adrenocorticalsuppressants (e.g., mitotane (o,p′-DDD) and aminoglutethimide); 11)adrenocorticosteroids (e.g., prednisone); 12) progestins (e.g.,hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrolacetate); 13) estrogens (e.g., diethylstilbestrol and ethinylestradiol); 14) antiestrogens (e.g., tamoxifen); 15) androgens (e.g.,testosterone propionate and fluoxymesterone); 16) antiandrogens (e.g.,flutamide): and 17) gonadotropin-releasing hormone analogs (e.g.,leuprolide).

Any oncolytic agent that is routinely used in a cancer therapy contextfinds use in the compositions and methods of the present disclosure. Forexample, the U.S. Food and Drug Administration maintains a formulary ofoncolytic agents approved for use in the United States. Internationalcounterpart agencies to the U.S.F.D.A. maintain similar formularies.Table 1 provides a list of exemplary antineoplastic agents approved foruse in the U.S. Those skilled in the art will appreciate that the“product labels” required on all U.S. approved chemotherapeuticsdescribe approved indications, dosing information, toxicity data, andthe like, for the exemplary agents.

TABLE 1 Aldesleukin Proleukin (des-alanyl-1, serine-125 humaninterleukin-2) Alemtuzumab Campath (IgG1κ anti CD52 antibody)Alitretinoin Panretin (9-cis-retinoic acid) Allopurinol Zyloprim(1,5-dihydro-4 H-pyrazolo[3,4-d]pyrimidin-4-one monosodium salt)Altretamine Hexalen (N,N,N′,N′,N″,N″,-hexamethyl-1,3,5-triazine-2,4,6-triamine) Amifostine Ethyol (ethanethiol, 2-[(3-aminopropyl)amino]-,dihydrogen phosphate (ester)) Anastrozole Arimidex(1,3-Benzenediacetonitrile, a,a,a′,a′-tetramethyl-5-(1H-1,2,4-triazol-1-ylmethyl)) Arsenic trioxide Trisenox Asparaginase Elspar(L-asparagine amidohydrolase, type EC-2) BCG Live TICE BCG (lyophilizedpreparation of an attenuated strain of Mycobacterium bovis (BacillusCalmette-Gukin [BCG], substrain Montreal) bexarotene capsules Targretin(4-[1-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2- napthalenyl) ethenyl]benzoic acid) bexarotene gel Targretin Bleomycin Blenoxane (cytotoxicglycopeptide antibiotics produced by Streptomyces verticillus; bleomycinA₂ and bleomycin B₂) Capecitabine Xeloda(5′-deoxy-5-fluoro-N-[(pentyloxy)carbonyl]-cytidine) CarboplatinParaplatin (platinum, diammine [1,1-cyclobutanedicarboxylato(2-)-0,0′]-, (SP-4-2)) Carmustine BCNU,(1,3-bis(2-chloroethyl)-1-nitrosourea) BiCNU Carmustine withPolifeprosan 20 Implant Gliadel Wafer Celecoxib Celebrex (as4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H- pyrazol-1-yl]benzenesulfonamide) Chlorambucil Leukeran(4-[bis(2chlorethyl)amino]benzenebutanoic acid) Cisplatin Platinol(PtCl₂H₆N₂) Cladribine Leustatin, (2-chloro-2′-deoxy-b-D-adenosine)2-CdA Cyclophosphamide Cytoxan, (2-[bis(2-chloroethyl)amino]tetrahydro-2H-13,2- Neosar oxazaphosphorine 2-oxide monohydrate)Cytarabine Cytosar-U (1-b-D-Arabinofuranosylcytosine, C₉H₁₃N₃O₅)cytarabine liposomal DepoCyt Dacarbazine DTIC-Dome(5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide (DTIC))Dactinomycin, actinomycin D Cosmegen (actinomycin produced byStreptomyces parvullus, C₆₂H₈₆N₁₂O₁₆) Darbepoetin alfa Aranesp(recombinant peptide) daunorubicin liposomal DanuoXome((8S-cis)-8-acetyl-10-[(3-amino-2,3,6-trideoxy-á-L-lyxo-hexopyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-methoxy-5,12-naphthacenedione hydrochloride)Daunorubicin HCl, daunomycin Cerubidine((1S,3S)-3-Acetyl-1,2,3,4,6,11-hexahydro-3,5,12-trihydroxy-10-methoxy-6,11-dioxo-1-naphthacenyl 3-amino-2,3,6-trideoxy-(alpha)-L-lyxo-hexopyranoside hydrochloride)Denileukin diftitox Ontak (recombinant peptide) Dexrazoxane Zinecard((S)-4,4′-(1-methyl-1,2-ethanediyl)bis-2,6- piperazinedione) DocetaxelTaxotere ((2R,3S)-N-carboxy-3-phenylisoserine, N-tert-butyl ester,13-ester with 5b-20-epoxy-12a,4,7b,10b,13a- hexahydroxytax-11-en-9-one4-acetate 2-benzoate, trihydrate) Doxorubicin HCl Adriamycin,(8S,10S)-10-[(3-amino-2,3,6-trideoxy-a-L-lyxo- Rubexhexopyranosyl)oxy]-8-glycolyl-7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-methoxy-5,12-naphthacenedione hydrochloride)doxorubicin Adriamycin PFS Intravenous injection doxorubicin liposomalDoxil dromostanolone propionate Dromostanolone(17b-Hydroxy-2a-methyl-5a-androstan-3-one propionate) dromostanolonepropionate Masterone injection Elliott's B Solution Elliott's B SolutionEpirubicin Ellence ((8S-cis)-10-[(3-amino-2,3,6-trideoxy-a-L-arabino-hexopyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12- naphthacenedionehydrochloride) Epoetin alfa Epogen (recombinant peptide) EstramustineEmcyt (estra-1,3,5(10)-triene-3,17-diol(17(beta))-, 3-[bis(2-chloroethyl)carbamate] 17-(dihydrogen phosphate), disodium salt,monohydrate, or estradiol 3-[bis(2- chloroethyl)carbamate]17-(dihydrogen phosphate), disodium salt, monohydrate) Etoposidephosphate Etopophos (4′-Demethylepipodophyllotoxin 9-[4,6-O-(R)-ethylidene-(beta)-D-glucopyranoside], 4′-(dihydrogen phosphate))etoposide, VP-16 Vepesid (4′-demethylepipodophyllotoxin 9-[4,6-0-(R)-ethylidene-(beta)-D-glucopyranoside]) Exemestane Aromasin(6-methylenandrosta-1,4-diene-3,17-dione) Filgrastim Neupogen(r-metHuG-CSF) floxuridine (intraarterial) FUDR(2′-deoxy-5-fluorouridine) Fludarabine Fludara (fluorinated nucleotideanalog of the antiviral agent vidarabine, 9-b-D-arabinofuranosyladenine(ara-A)) Fluorouracil, 5-FU Adrucil(5-fluoro-2,4(1H,3H)-pyrimidinedione) Fulvestrant Faslodex(7-alpha-[9-(4,4,5,5,5-penta fluoropentylsulphinyl)nonyl]estra-1,3,5-(10)-triene-3,17-beta-diol) Gemcitabine Gemzar(2′-deoxy-2′,2′-difluorocytidine monohydrochloride (b- isomer))Gemtuzumab Ozogamicin Mylotarg (anti-CD33 hP67.6) Goserelin acetateZoladex Implant Hydroxyurea Hydrea Ibritumomab Tiuxetan Zevalin(immunoconjugate resulting from a thiourea covalent bond between themonoclonal antibody Ibritumomab and the linker-chelator tiuxetan [N-[2-bis(carboxymethyl)amino]-3-(p-isothiocyanatophenyl)-propyl]-[N-[2-bis(carboxymethyl)amino]-2-(methyl)- ethyl]glycine)Idarubicin Idamycin (5,12-Naphthacenedione, 9-acetyl-7-[(3-amino-2,3,6-trideoxy-(alpha)-L-lyxo-hexopyranosyl)oxy]-7,8,9,10-tetrahydro-6,9,11-trihydroxyhydrochloride, (7S-cis)) Ifosfamide IFEX(3-(2-chloroethyl)-2-[(2-chloroethyl)amino]tetrahydro-2H-1,3,2-oxazaphosphorine 2-oxide) Imatinib Mesilate Gleevec(4-[(4-Methyl-1-piperazinyl)methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]- phenyl]benzamidemethanesulfonate) Interferon alfa-2a Roferon-A (recombinant peptide)Interferon alfa-2b Intron A (recombinant peptide) (LyophilizedBetaseron) Irinotecan HCl Camptosar ((4S)-4,11-diethyl-4-hydroxy-9-[(4-piperidinopiperidino)carbonyloxy]-1H-pyrano[3′,4′: 6,7]indolizino[1,2-b] quinoline-3,14(4H,12H) dione hydrochloride trihydrate)Letrozole Femara (4,4′-(1H-1,2,4-Triazol-1-ylmethylene) dibenzonitrile)Leucovorin Wellcovorin, (L-Glutamic acid,N[4[[(2amino-5-formyl-1,4,5,6,7,8 Leucovorinhexahydro4oxo6-pteridinyl)methyl]amino]benzoyl], calcium salt (1:1))Levamisole HCl Ergamisol ((−)-(S)-2,3,5,6-tetrahydro-6-phenylimidazo[2,1-b] thiazole monohydrochloride C₁₁H₁₂N₂S•HCl) Lomustine CeeNU(1-(2-chloro-ethyl)-3-cyclohexyl-1-nitrosourea) Meclorethamine, nitrogenmustard Mustargen (2-chloro-N-(2-chloroethyl)-N-methylethanaminehydrochloride) Megestrol acetate Megace17α(acetyloxy)-6-methylpregna-4,6-diene-3,20-dione Melphalan, L-PAMAlkeran (4-[bis(2-chloroethyl) amino]-L-phenylalanine) Mercaptopurine,6-MP Purinethol (1,7-dihydro-6 H-purine-6-thione monohydrate) MesnaMesnex (sodium 2-mercaptoethane sulfonate) Methotrexate Methotrexate(N-[4-[[(2,4-diamino-6-pteridinyl)methyl]methylamino]benzoyl]-L-glutamic acid) MethoxsalenUvadex (9-methoxy-7H-furo[3,2-g][1]-benzopyran-7-one) Mitomycin CMutamycin mitomycin C Mitozytrex Mitotane Lysodren(1,1-dichloro-2-(o-chlorophenyl)-2-(p-chlorophenyl) ethane) MitoxantroneNovantrone (1,4-dihydroxy-5,8-bis[[2-[(2-hydroxyethyl)amino]ethyl]amino]-9,10-anthracene- dione dihydrochloride)Nandrolone phenpropionate Durabolin-50 Nofetumomab Verluma OprelvekinNeumega (IL-11) Oxaliplatin Eloxatin(cis-[(1R,2R)-1,2-cyclohexanediamine-N,N′] [oxalato(2-)-O,O′] platinum)Paclitaxel TAXOL (5β,20-Epoxy-1,2a,4,7β,10β,13a-hexahydroxytax-11-en-9-one 4,10-diacetate 2-benzoate 13-ester with(2R,3S)-N-benzoyl-3-phenylisoserine) Pamidronate Aredia (phosphonic acid(3-amino-1-hydroxypropylidene) bis-, disodium salt, pentahydrate, (APD))Pegademase Adagen ((monomethoxypolyethylene glycol succinimidyl) 11-(Pegademase 17-adenosine deaminase) Bovine) Pegaspargase Oncaspar(monomethoxypolyethylene glycol succinimidyl L-asparaginase)Pegfilgrastim Neulasta (covalent conjugate of recombinant methionylhuman G-CSF (Filgrastim) and monomethoxypolyethylene glycol) PentostatinNipent Pipobroman Vercyte Plicamycin, Mithramycin Mithracin (antibioticproduced by Streptomyces plicatus) Porfimer sodium PhotofrinProcarbazine Matulane (N-isopropyl-μ-(2-methylhydrazino)-p-toluamidemonohydrochloride) Quinacrine Atabrine(6-chloro-9-(1-methyl-4-diethyl-amine) butylamino-2- methoxyacridine)Rasburicase Elitek (recombinant peptide) Rituximab Rituxan (recombinantanti-CD20 antibody) Sargramostim Prokine (recombinant peptide)Streptozocin Zanosar (streptozocin 2-deoxy-2-[[(methylnitrosoamino)carbonyl]amino]-a(and b)-D- glucopyranose and 220mg citric acid anhydrous) Talc Sclerosol (Mg₃Si₄O₁₀ (OH)₂) TamoxifenNolvadex ((Z)2-[4-(1,2-diphenyl-1-butenyl) phenoxy]-N,N-dimethylethanamine 2-hydroxy-1,2,3- propanetricarboxylate (1:1))Temozolomide Temodar (3,4-dihydro-3-methyl-4-oxoimidazo[5,1-d]-as-tetrazine-8-carboxamide) teniposide, VM-26 Vumon(4′-demethylepipodophyllotoxin 9-[4,6-0-(R)-2-thenylidene-(beta)-D-glucopyranoside]) Testolactone Teslac(13-hydroxy-3-oxo-13,17-secoandrosta-1,4-dien-17-oic acid [dgr]-lactone)Thioguanine, 6-TG Thioguanine (2-amino-1,7-dihydro-6 H-purine-6-thione)Thiotepa Thioplex (Aziridine,1,1′,1″-phosphinothioylidynetris-, or Tris(1-aziridinyl) phosphine sulfide) Topotecan HCl Hycamtin((S)-10-[(dimethylamino) methyl]-4-ethyl-4,9- dihydroxy-1H-pyrano[3′,4′:6,7] indolizino [1,2-b] quinoline-3,14-4H,12H)-dione monohydrochloride)Toremifene Fareston(2-(p-[(Z)-4-chloro-1,2-diphenyl-1-butenyl]-phenoxy)-N,N-dimethylethylamine citrate (1:1)) Tositumomab, I 131 TositumomabBexxar (recombinant murine immunotherapeutic monoclonal IgG_(2a) lambdaanti-CD20 antibody (I 131 is a radioimmunotherapeutic antibody))Trastuzumab Herceptin (recombinant monoclonal IgG₁ kappa anti-HER2antibody) Tretinoin, ATRA Vesanoid (all-trans retinoic acid) UracilMustard Uracil Mustard Capsules Valrubicin,N-trifluoroacetyladriamycin-14-valerate Valstar((2S-cis)-2-[1,2,3,4,6,11-hexahydro-2,5,12-trihydroxy- 7methoxy-6,11-dioxo-[[4 2,3,6-trideoxy-3-[(trifluoroacetyl)-amino-α-L-lyxo-hexopyranosyl]oxyl]-2-naphthacenyl]-2- oxoethyl pentanoate) Vinblastine,Leurocristine Velban (C₄₆H₅₆N₄O₁₀•H₂SO₄) Vincristine Oncovin(C₄₆H₅₆N₄O₁₀•H₂SO₄) Vinorelbine Navelbine(3′,4′-didehydro-4′-deoxy-C′-norvincaleukoblastine [R-(R*,R*)-2,3-dihydroxybutanedioate (1:2)(salt)]) Zoledronate, Zoledronicacid Zometa ((1-Hydroxy-2-imidazol-1-yl-phosphonoethyl) phosphonic acidmonohydrate)

Anticancer agents further include compounds which have been identifiedto have anticancer activity. Examples include, but are not limited to,3-AP, 12-O-tetradecanoylphorbol-13-acetate, 17AAG, 852A, ABI-007,ABR-217620, ABT-751, ADI-PEG 20, AE-941, AG-013736, AGRO100, alanosine,AMG 706, antibody G250, antineoplastons, AP23573, apaziquone, APC8015,atiprimod, ATN-161, atrasenten, azacitidine, BB-10901, BCX-1777,bevacizumab, BG00001, bicalutamide, BMS 247550, bortezomib,bryostatin-1, buserelin, calcitriol, CCI-779, CDB-2914, cefixime,cetuximab, CG0070, cilengitide, clofarabine, combretastatin A4phosphate, CP-675,206, CP-724,714, CpG 7909, curcumin, decitabine,DENSPM, doxercalciferol, E7070, E7389, ecteinascidin 743, efaproxiral,eflornithine, EKB-569, enzastaurin, erlotinib, exisulind, fenretinide,flavopiridol, fludarabine, flutamide, fotemustine, FR901228, G17DT,galiximab, gefitinib, genistein, glufosfamide, GTI-2040, histrelin,HKI-272, homoharringtonine, HSPPC-96, hu14.18-interleukin-2 fusionprotein, HuMax-CD4, iloprost, imiquimod, infliximab, interleukin-12,IPI-504, irofulven, ixabepilone, lapatinib, lenalidomide, lestaurtinib,leuprolide, LMB-9 immunotoxin, lonafarnib, luniliximab, mafosfamide,MB07133, MDX-010, MLN2704, monoclonal antibody 3F8, monoclonal antibodyJ591, motexafin, MS-275, MVA-MUC1-IL2, nilutamide, nitrocamptothecin,nolatrexed dihydrochloride, nolvadex, NS-9,06-benzylguanine, oblimersensodium, ONYX-015, oregovomab, OSI-774, panitumumab, paraplatin,PD-0325901, pemetrexed, PHY906, pioglitazone, pirfenidone, pixantrone,PS-341, PSC 833, PXD101, pyrazoloacridine, R115777, RAD001, ranpirnase,rebeccamycin analogue, rhuAngiostatin protein, rhuMab 2C4,rosiglitazone, rubitecan, S-1, S-8184, satraplatin, SB-, 15992,SGN-0010, SGN-40, sorafenib, SR31747A, ST1571, SU011248, suberoylanilidehydroxamic acid, suramin, talabostat, talampanel, tariquidar,temsirolimus, TGFa-PE38 immunotoxin, thalidomide, thymalfasin,tipifarnib, tirapazamine, TLK286, trabectedin, trimetrexate glucuronate,TroVax, UCN-1, valproic acid, vinflunine, VNP40101M, volociximab,vorinostat, VX-680, ZD1839, ZD6474, zileuton, and zosuquidartrihydrochloride.

For a more detailed description of anticancer agents and othertherapeutic agents, those skilled in the art are referred to any numberof instructive manuals including, but not limited to, the Physician'sDesk Reference and to Goodman and Gilman's “Pharmaceutical Basis ofTherapeutics” tenth edition, Eds. Hardman et al., 2002.

In some embodiments, methods provided herein comprise administering oneor more compounds provided herein with radiation therapy. The methodsprovided herein are not limited by the types, amounts, or delivery andadministration systems used to deliver the therapeutic dose of radiationto an animal. For example, the animal may receive photon radiotherapy,particle beam radiation therapy, other types of radiotherapies, andcombinations thereof. In some embodiments, the radiation is delivered tothe animal using a linear accelerator. In still other embodiments, theradiation is delivered using a gamma knife.

The source of radiation can be external or internal to the animal.External radiation therapy is most common and involves directing a beamof high-energy radiation to a tumor site through the skin using, forinstance, a linear accelerator. While the beam of radiation is localizedto the tumor site, it is nearly impossible to avoid exposure of normal,healthy tissue. However, external radiation is usually well tolerated byanimals. Internal radiation therapy involves implanting aradiation-emitting source, such as beads, wires, pellets, capsules,particles, and the like, inside the body at or near the tumor siteincluding the use of delivery systems that specifically target cancercells (e.g., using particles attached to cancer cell binding ligands).Such implants can be removed following treatment, or left in the bodyinactive. Types of internal radiation therapy include, but are notlimited to, brachytherapy, interstitial irradiation, intracavityirradiation, radioimmunotherapy, and the like.

The animal may optionally receive radiosensitizers (e.g., metronidazole,misonidazole, intra-arterial Budr, intravenous iododeoxyuridine (IudR),nitroimidazole, 5-substituted-4-nitroimidazoles, 2H-isoindolediones,[[(2-bromoethyl)-amino]methyl]-nitro-1H-imidazole-1-ethanol,nitroaniline derivatives, DNA-affinic hypoxia selective cytotoxins,halogenated DNA ligand, 1,2,4 benzotriazine oxides, 2-nitroimidazolederivatives, fluorine-containing nitroazole derivatives, benzamide,nicotinamide, acridine-intercalator, 5-thiotretrazole derivative,3-nitro-1,2,4-triazole, 4,5-dinitroimidazole derivative, hydroxylatedtexaphrins, cisplatin, mitomycin, tiripazamine, nitrosourea,mercaptopurine, methotrexate, fluorouracil, bleomycin, vincristine,carboplatin, epirubicin, doxorubicin, cyclophosphamide, vindesine,etoposide, paclitaxel, heat (hyperthermia), and the like),radioprotectors (e.g., cysteamine, aminoalkyl dihydrogenphosphorothioates, amifostine (WR 2721), IL-1, IL-6, and the like).Radiosensitizers enhance the killing of tumor cells. Radioprotectorsprotect healthy tissue from the harmful effects of radiation.

Any type of radiation can be administered to an animal, so long as thedose of radiation is tolerated by the animal without unacceptablenegative side-effects. Suitable types of radiotherapy include, forexample, ionizing (electromagnetic) radiotherapy (e.g., X-rays or gammarays) or particle beam radiation therapy (e.g., high linear energyradiation). Ionizing radiation is defined as radiation comprisingparticles or photons that have sufficient energy to produce ionization,i.e., gain or loss of electrons (as described in, for example, U.S. Pat.No. 5,770,581 incorporated herein by reference in its entirety). Theeffects of radiation can be at least partially controlled by theclinician. In one embodiment, the dose of radiation is fractionated formaximal target cell exposure and reduced toxicity.

In one embodiment, the total dose of radiation administered to an animalis about 0.01 Gray (Gy) to about 100 Gy. In another embodiment, about 10Gy to about 65 Gy (e.g., about 15 Gy, 20 Gy, 25 Gy, 30 Gy, 35 Gy, 40 Gy,45 Gy, 50 Gy, 55 Gy, or 60 Gy) are administered over the course oftreatment. While in some embodiments a complete dose of radiation can beadministered over the course of one day, the total dose is ideallyfractionated and administered over several days. Desirably, radiotherapyis administered over the course of at least about 3 days, e.g., at least5, 7, 10, 14, 17, 21, 25, 28, 32, 35, 38, 42, 46, 52, or 56 days (about1-8 weeks). Accordingly, a daily dose of radiation will compriseapproximately 1-5 Gy (e.g., about 1 Gy, 1.5 Gy, 1.8 Gy, 2 Gy, 2.5 Gy,2.8 Gy, 3 Gy, 3.2 Gy, 3.5 Gy, 3.8 Gy, 4 Gy, 4.2 Gy, or 4.5 Gy), or 1-2Gy (e.g., 1.5-2 Gy). The daily dose of radiation should be sufficient toinduce destruction of the targeted cells. If stretched over a period, inone embodiment, radiation is not administered every day, therebyallowing the animal to rest and the effects of the therapy to berealized. For example, radiation desirably is administered on 5consecutive days, and not administered on 2 days, for each week oftreatment, thereby allowing 2 days of rest per week. However, radiationcan be administered 1 day/week, 2 days/week, 3 days/week, 4 days/week, 5days/week, 6 days/week, or all 7 days/week, depending on the animal'sresponsiveness and any potential side effects. Radiation therapy can beinitiated at any time in the therapeutic period. In one embodiment,radiation is initiated in week 1 or week 2, and is administered for theremaining duration of the therapeutic period. For example, radiation isadministered in weeks 1-6 or in weeks 2-6 of a therapeutic periodcomprising 6 weeks for treating, for instance, a solid tumor.Alternatively, radiation is administered in weeks 1-5 or weeks 2-5 of atherapeutic period comprising 5 weeks. These exemplary radiotherapyadministration schedules are not intended, however, to limit the methodsprovided herein.

Antimicrobial therapeutic agents may also be used as therapeutic agentsin combination with the compounds provided herein. Any agent that cankill, inhibit, or otherwise attenuate the function of microbialorganisms may be used, as well as any agent contemplated to have suchactivities. Antimicrobial agents include, but are not limited to,natural and synthetic antibiotics, antibodies, inhibitory proteins(e.g., defensins), antisense nucleic acids, membrane disruptive agentsand the like, used alone or in combination. Indeed, any type ofantibiotic may be used including, but not limited to, antibacterialagents, antiviral agents, antifungal agents, and the like.

In some embodiments of the methods provided herein, one or morecompounds provided herein and one or more therapeutic agents oranticancer agents are administered to an animal under one or more of thefollowing conditions: at different periodicities, at differentdurations, at different concentrations, by different administrationroutes, etc. In some embodiments, the compound is administered prior tothe therapeutic or anticancer agent, e.g., 0.5, 1, 2, 3, 4, 5, 10, 12,or 18 hours, 1, 2, 3, 4, 5, or 6 days, or 1, 2, 3, or 4 weeks prior tothe administration of the therapeutic or anticancer agent. In someembodiments, the compound is administered after the therapeutic oranticancer agent, e.g., 0.5, 1, 2, 3, 4, 5, 10, 12, or 18 hours, 1, 2,3, 4, 5, or 6 days, or 1, 2, 3, or 4 weeks after the administration ofthe anticancer agent. In some embodiments, the compound and thetherapeutic or anticancer agent are administered concurrently but ondifferent schedules, e.g., the compound is administered daily while thetherapeutic or anticancer agent is administered once a week, once everytwo weeks, once every three weeks, or once every four weeks. In otherembodiments, the compound is administered once a week while thetherapeutic or anticancer agent is administered daily, once a week, onceevery two weeks, once every three weeks, or once every four weeks.

In some embodiments, compositions provided herein comprise one or moreof the compounds provided herein in an amount which is effective toachieve its intended purpose. While individual needs vary, determinationof optimal ranges of effective amounts of each component is within theskill of the art. Typically, the compounds may be administered tomammals, e.g. humans, orally at a dose of 0.0025 to 50 mg/kg, or anequivalent amount of the pharmaceutically acceptable salt thereof, perday of the body weight of the mammal being treated for disordersresponsive to induction of apoptosis. In one embodiment, about 0.01 toabout 25 mg/kg is orally administered to treat, ameliorate, or preventsuch disorders. For intramuscular injection, the dose is generally aboutone-half of the oral dose. For example, a suitable intramuscular dosewould be about 0.0025 to about 25 mg/kg, or from about 0.01 to about 5mg/kg.

The unit oral dose may comprise from about 0.01 to about 1000 mg, forexample, about 0.1 to about 100 mg of the compound. The unit dose may beadministered one or more times daily as one or more tablets or capsuleseach containing from about 0.1 to about 10 mg, conveniently about 0.25to 50 mg of the compound or its solvates.

In a topical formulation, the compound may be present at a concentrationof about 0.01 to 100 mg per gram of carrier. In a one embodiment, thecompound is present at a concentration of about 0.07-1.0 mg/ml, forexample, about 0.1-0.5 mg/ml, and in one embodiment, about 0.4 mg/ml.

In addition to administering the compound as a raw chemical, thecompounds provided herein may be administered as part of apharmaceutical preparation. In some embodiments, the pharmaceuticalpreparation can include one or more pharmaceutically acceptable carrier,excipient, and/or auxiliary. In some embodiments, the one or morecarriers, excipients, and auxiliaries facilitate processing of thecompound into a preparation which can be used pharmaceutically. Thepreparations, particularly those preparations which can be administeredorally or topically and which can be used for one type ofadministration, such as tablets, dragees, slow release lozenges andcapsules, mouth rinses and mouth washes, gels, liquid suspensions, hairrinses, hair gels, shampoos and also preparations which can beadministered rectally, such as suppositories, as well as suitablesolutions for administration by intravenous infusion, injection,topically or orally, contain from about 0.01 to 99 percent, in oneembodiment from about 0.25 to 75 percent of active compound(s), togetherwith the one or more carriers, excipients, and/or auxiliaries.

The pharmaceutical compositions of provided herein may be administeredto any patient which may experience the beneficial effects of thecompounds provided herein. Foremost among such patients are mammals,e.g., humans, although the methods and compositions provided herein arenot intended to be so limited. Other patients include veterinary animals(cows, sheep, pigs, horses, dogs, cats and the like).

The compounds and pharmaceutical compositions thereof may beadministered by any means that achieve their intended purpose. Forexample, administration may be by parenteral, subcutaneous, intravenous,intramuscular, intraperitoneal, transdermal, buccal, intrathecal,intracranial, intranasal or topical routes. Alternatively, orconcurrently, administration may be by the oral route. The dosageadministered will be dependent upon the age, health, and weight of therecipient, kind of concurrent treatment, if any, frequency of treatment,and the nature of the effect desired.

The pharmaceutical preparations provided herein are manufactured bymeans of conventional mixing, granulating, dragee-making, dissolving, orlyophilizing processes. Thus, pharmaceutical preparations for oral usecan be obtained by combining the active compounds with solid excipients,optionally grinding the resulting mixture and processing the mixture ofgranules, after adding suitable auxiliaries, if desired or necessary, toobtain tablets or dragee cores.

Suitable excipients are, in particular, fillers such as saccharides, forexample lactose or sucrose, mannitol or sorbitol, cellulose preparationsand/or calcium phosphates, for example tricalcium phosphate or calciumhydrogen phosphate, as well as binders such as starch paste, using, forexample, maize starch, wheat starch, rice starch, potato starch,gelatin, tragacanth, methyl cellulose, hydroxypropylmethylcellulose,sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone. If desired,disintegrating agents may be added such as the above-mentioned starchesand also carboxymethyl-starch, cross-linked polyvinyl pyrrolidone, agar,or alginic acid or a salt thereof, such as sodium alginate. Auxiliariescan be suitable flow-regulating agents and lubricants. Suitableauxiliaries include, for example, silica, talc, stearic acid or saltsthereof, such as magnesium stearate or calcium stearate, and/orpolyethylene glycol. Dragee cores are provided with suitable coatingswhich, if desired, are resistant to gastric juices. For this purpose,concentrated saccharide solutions may be used, which may optionallycontain gum arabic, talc, polyvinyl pyrrolidone, polyethylene glycoland/or titanium dioxide, lacquer solutions and suitable organic solventsor solvent mixtures. In order to produce coatings resistant to gastricjuices, solutions of suitable cellulose preparations such asacetylcellulose phthalate or hydroxypropylmethyl-cellulose phthalate,are used. Dye stuffs or pigments may be added to the tablets or drageecoatings, for example, for identification or in order to characterizecombinations of active compound doses.

Other pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer such as glycerol or sorbitol. The push-fitcapsules can contain the active compounds in the form of granules whichmay be mixed with fillers such as lactose, binders such as starches,and/or lubricants such as talc or magnesium stearate and, optionally,stabilizers. In soft capsules, the active compounds are in oneembodiment dissolved or suspended in suitable liquids, such as fattyoils, or liquid paraffin. In addition, stabilizers may be added.

Possible pharmaceutical preparations which can be used rectally include,for example, suppositories, which consist of a combination of one ormore of the active compounds with a suppository base. Suitablesuppository bases are, for example, natural or synthetic triglycerides,or paraffin hydrocarbons. In addition, it is also possible to usegelatin rectal capsules which consist of a combination of the activecompounds with a base. Possible base materials include, for example,liquid triglycerides, polyethylene glycols, or paraffin hydrocarbons.

Suitable formulations for parenteral administration include aqueoussolutions of the active compounds in water-soluble form, for example,water-soluble salts and alkaline solutions. In addition, suspensions ofthe active compounds as appropriate oily injection suspensions may beadministered. Suitable lipophilic solvents or vehicles include fattyoils, for example, sesame oil, or synthetic fatty acid esters, forexample, ethyl oleate or triglycerides or polyethylene glycol-400.Aqueous injection suspensions may contain substances which increase theviscosity of the suspension include, for example, sodium carboxymethylcellulose, sorbitol, and/or dextran. Optionally, the suspension may alsocontain stabilizers.

The topical compositions provided herein are formulated in oneembodiment as oils, creams, lotions, ointments and the like by choice ofappropriate carriers. Suitable carriers include vegetable or mineraloils, white petrolatum (white soft paraffin), branched chain fats oroils, animal fats and high molecular weight alcohol (greater than C₁₂).The carriers may be those in which the active ingredient is soluble.Emulsifiers, stabilizers, humectants and antioxidants may also beincluded as well as agents imparting color or fragrance, if desired.Additionally, transdermal penetration enhancers can be employed in thesetopical formulations. Examples of such enhancers can be found in U.S.Pat. Nos. 3,989,816 and 4,444,762.

Ointments may be formulated by mixing a solution of the activeingredient in a vegetable oil such as almond oil with warm soft paraffinand allowing the mixture to cool. A typical example of such an ointmentis one which includes about 30% almond oil and about 70% white softparaffin by weight. Lotions may be conveniently prepared by dissolvingthe active ingredient, in a suitable high molecular weight alcohol suchas propylene glycol or polyethylene glycol.

The following examples are illustrative, but not limiting, of thecompounds, compositions, and methods provided herein. Other suitablemodifications and adaptations of the variety of conditions andparameters normally encountered in clinical therapy and which areobvious to those skilled in the art are within the spirit and scope ofthe methods, compounds, and compositions provided herein.

MDM2 Inhibitors

U.S. Pat. Nos. 7,759,383 B2 and 7,737,174 B2 disclose MDM2 inhibitorsincluding MI-219 (AT-219), MI-319 and MI-147 (Chart 1).

To further evaluate MI-219, MI-147, and MI-319 as potential anticancerdrug candidates for clinical development, a series of experiments werecarried out to evaluate their metabolic and pharmacokinetic properties.

Incubation of MI-219, MI-147 and MI-319 with human, rat and dogmicrosomes in vitro showed that the concentration of each of these threecompounds decreased rather quickly (Table 2). These data suggested thatMI-219, MI-147 and MI-319 are metabolized fairly rapidly in vitro byrat, dog and human microsomes.

Pharmacokinetic studies (PK) of MI-219 in Male Sprague Dawley ratsshowed that MI-219 was orally bioavailable (Tables 3-4). For example,following oral administration of MI-219 at dose of 25 mg/kg, the mean±SDvalues of Cmax(maximum plasma concentration) and Tmax for MI-219 were3751.78±1067.86 μg/L, 0.58±0.38 hr, respectively; the mean±SD values ofAUC(0-∞) (area-under-the-curve) and half-life (T½) for MI-219 were7689.94±325.86 hr*μg/L and 1.43±0.09 hr, respectively. The plasmaconcentrations of MI-219 decreased fairly rapidly, from 2957 ng/ml at 1hr time-point, to 224 ng/ml at 4 hr time-point, further to 103 ng/ml at6 hr time-point. The calculated oral bioavailability in rats for MI-219using the 25 mg/kg oral dosing and 5 mg/kg IV dosing as the referenceswas 65.45±2.77%.

PK studies of MI-219 in ICR mice showed that MI-219 was orallybioavailable in mice (Tables 5-7). At 50 mg/kg oral dosing, MI-219reached the maximum concentration (Cmax) of 8469 ng/ml at 2 hrtime-point and had an AUC of 8469±2381 hr*ug/L. The concentrations ofMI-219 were 3077±2296, 378±128 and 121±133 ng/ml at 3 h, 5 h and 9 h,respectively. The calculated oral bioavailability (F) for MI-219 in miceis 54.9±19.0%.

PK studies of MI-219 in Beagle dogs showed that MI-219 was orallybioavailable in dogs (Tables 8-11). At 10 mg/kg oral dosing, MI-219reached the maximum concentration (Cmax) of 2893±726 ng/ml at 1 hrtime-point and had an AUC of 7947±2396 hr*ug/L. The concentrations ofMI-219 were 1500±599, 759±314, 483±152, 327±147, 178±92 ng/ml at 2, 3,4, 6 and 8 hr time-point, respectively. The calculated oralbioavailability (F) for MI-219 in dogs is 22.8%.

PK studies of MI-219 in Cynomolgus Monkeys showed that MI-219 was orallybioavailable in monkeys (Tables 12-13). At 50 mg/kg oral dosing, MI-219reached the maximum concentration (Cmax) of 1257±837 at 3.3 hr by curvefitting and had an AUC of 8199±5717 hr*ug/L. The concentrations ofMI-219 were 1121.42±951.73, 796.07±703.14, 341.82±273.89 and 17.60±7.21at 4, 6, 8 and 24 hr time-point, respectively. The calculated oralbioavailability (F) of MI-219 in moneys is 13.89%.

Oral administration of MI-219 in mice bearing SJSA-1 and LnCAP humanxenograft tumors was indeed effective tumor growth inhibition at 200-300mg/kg daily dosing or twice a day for a period of 2 weeks (Shangary etal. 2008, PNAS). SJSA-1 cells are derived from the primary tumor or apatient diagnosed with primitive multipotential sarcoma of the femur.SJSA-1 cells harbor an amplification of the MDM2 gene. LnCAP cells areandrogen-sensitive human prostate adenocarcinoma cells derived from ametastatic tumor of a patient diagnosed with prostate cancer. These dataindicate that MI-219 is an orally active MDM2 inhibitor.

These metabolic and PK studies also revealed that MI-219 is metabolizedfairly quickly. Hence, further chemical modifications of MI-219 toimprove upon its metabolic and PK parameters may yield new MDM2inhibitors as better drug candidates for treatment of human cancer andother conditions through targeting human MDM2 and activation of p53.

Provided herein are new compounds that inhibit p53-MDM2 interaction. Thecompounds provided herein have, inter alia, improved metabolic stabilityand/or pharmacokinetic properties and oral bioavailability.

TABLE 2 Microsomal stability studies of MI-147, MI-219 and MI-319. Time(min) MI-147 MI-219 MI-319 Human 0 100.00% 100.00% 100.00% 5 78.23%78.76% 82.09% 10 58.01% 58.19% 60.88% 15 41.59% 46.46% 43.86% 30 19.67%26.46% 24.79% 45 10.81% 20.88% 10.66% 60 7.18% 11.86% 16.31% Rat 0100.00% 100.00% 100.00% 5 68.05% 58.19% 64.80% 10 31.04% 40.59% 41.96%15 16.85% 28.11% 30.71% 30 6.18% 18.61% 23.11% 45 4.40% 18.42% 23.01% 603.43% 18.38% 24.01% Dog 0 100.00% 100.00% 100.00% 5 77.84% 83.38%108.25% 10 47.98% 71.22% 69.29% 15 44.83% 71.51% 57.20% 30 21.52% 54.96%41.84% 45 17.08% 33.81% 29.39% 60 12.15% 32.52% 27.06% Negative 0100.00% 100.00% 100.00% 5 94.73% 102.63% 95.37% 10 90.77% 105.25% 90.75%15 93.43% 109.19% 96.26% 30 94.14% 111.07% 86.12% 45 91.07% 104.13%91.41% 60 85.38% 109.01% 85.90%

TABLE 3 Plasma Concentration of MI-219 in Male Rat Following Intravenousand Oral Administration. Time Point (hr) Plasma Concentration (ng/mL)Animal IV (5 mg/kg) PO (25 mg/kg) No. M1 M2 Mean M6 M7 M8 Mean SD 0.0835873.30 5229.30 5551.30 1526.57 1380.03 1642.42 1516.34 131.49 0.251551.07 1612.79 1581.93 2817.28 2398.33 4965.7 3393.77 1377.35 0.5693.45 548.39 620.92 3332.19 2500.25 4278.56 3370.33 889.77 1 330.71344.66 337.68 3101.06 2957.44 1779.31 2612.60 725.22 2 164.88 134.91149.90 1064.21 1534.41 1342.18 1313.60 236.40 4 49.27 56.50 52.88296.644 223.937 407.415 309.33 92.39 6 24.18 32.66 28.42 103.285 90.8093131.398 108.50 20.79 8 11.05 18.98 15.02 47.5486 80.4445 66.2777 64.7616.50 24 1.86 1.28 1.57 25.1348 17.5136 20.2154 20.95 3.86 BLQ: Belowthe limit of quantitation; SD: Standard deviation; NA: Nonapplicable

TABLE 4 Selected Pharmacokinetics Parameters of MI-219 in Rats FollowingIntravenous and Oral Administration. Pharmacokinetic Parameters AnimalAUC_((0-t)) AUC_((0-∞)) MRT_((0-∞)) T_(1/2) Vz CLz Cmax Tmax F Numberhr * ug/L hr * ug/L hr hr L/kg L/hr/kg ug/L hr (%) IV (5 mg/kg) 12379.54 2379.62 1.26 1.86 5.62 2.10 5873.30 2 2318.88 2319.77 1.51 2.547.90 2.16 5229.30 MEAN 2349.21 2349.69 1.38 2.20 6.76 2.13 5551.30 PO(25 mg/kg) 6 7378.78 7378.81 2.64 1.36 NA NA 3332.19 0.5 62.81 7 7623.457662.25 2.67 1.54 NA NA 2957.44 1 65.22 8 8028.71 8028.76 2.62 1.40 NANA 4965.7 0.25 68.34 MEAN 7676.98 7689.94 2.64 1.43 NA NA 3751.78 0.5865.45 SD 328.26 325.86 0.03 0.09 NA NA 1067.86 0.38 2.77 NA:Nonapplicable

TABLE 5 Measured concentrations of MI-219 in the plasma samples after asingle p.o dose at 50 mg/kg in mice. Group 3-8 Group 3-10 Group 3-12Mean ± SD Time Plasma Concentrations (ng/ml) 5 min 944 295 1016 752 ±397 15 min 2927 1700 1503 2043 ± 772  30 min 2312 5956 4371 4213 ± 182745 min 3354 4737 9082 5724 ± 2988 1 h 9267 2693 4800 5587 ± 3357 2 h10399 9201 5808 8469 ± 2381 3 h 5719 1948 1564 3077 ± 2296 5 h 522 277334 378 ± 128 9 h 272 26 64.2 121 ± 133 24 h Beyond Beyond Beyonddetection detection detection limit limit limit

TABLE 6 Measured concentrations of MI-219 in the plasma samples after asingle i.v. dose at 10 mg/kg in mice. Time Group 4-7 Group 4-9 Group4-11 Mean ± SD 5 min 14196 21792 18135 18041 ± 3799 15 min 6030 63975042 5823 ± 701 30 min 2628 4347 3735 3570 ± 871 45 min 1519 2225 14491731 ± 429 1 h 1061 1285 1322 1223 ± 141 2 h 689 325 1328  781 ± 508 3 h108 489 150  249 ± 209 5 h 78.5 52 38.6  56.4 ± 20.3 9 h 13.4 37.2 13.9 21.5 ± 13.6 24 h BLQ BLQ BLQ

TABLE 7 PK parameters of MI-219 after a p.o. dose (50 mg/kg) in mice.Parameter Group 3-8 Group 3-10 Group 3-12 Mean ± SD Oral dose Cmax 103999201 9082 9561 ± 728  (ng/mL) Tmax (h) 2 2 0.75 1.58 ± 0.72 AUC 0→9 h26839 16158 14622 19206 ± 6655  (ng · h/mL) MRT (h) 2.5813 2.043072.2585 2.29 ± 0.27 Kel 0.37 0.69 0.35 0.47 ± 0.19 T½ 1.51 0.99 1.82 1.61± 0.53 F (%) 76.7 46.2 41.8 54.9 ± 19.0

TABLE 8 Plasma concentration of MI-219 following IV administration at2.0 mg/kg to Beagle dogs (ng/ml). Dog Time (h) No. 0.117 0.133 0.20 0.51.0 3.0 4.0 6.0 8.0 12 24 1 — 7464 2623 1888 853 367 256 114.3 52.2 17.93.55 2 4069 — 2272 1192 704 331 188 81.1 39.7 9.75 1.11 3 7668 — 39181795 1044 402 248 119 57.8 19.4 5.62 Mean 5868 7464 2938 1625 867 367231 105 49.9 15.7 3.42 SD 2545 867 378 170 36 37 21 9.3 5.2 2.26 CV %43.4 29.5 23.3 19.6 9.8 16.0 19.8 18.6 33.1 65.9

TABLE 9 Plasma concentration of MI-219 following PO administration at 10mg/kg to Beagle dogs (ng/ml). Dog Time (h) No. 0 0.5 1 2 3 4 6 8 12 24 1BLQ 2101 3560 2165 1095 603 335 216 54.68 4.05 2 BLQ 1842 2119 1002 474312 177 72.9 20.0 3.78 3 BLQ 3323 2999 1333 708 535 470 245 63.8 4.48Mean 2422 2893 1500 759 483 327 178 46.2 4.10 SD 791 726 599 314 152 14792 23.1 0.35 CV % 32.7 25.1 40.0 41.3 31.5 44.8 51.8 50.0 8.6 Min 18422119 1002 474 312 177 72.9 20.0 3.78 Max 3323 3560 2165 1095 603 470 24563.8 4.48

TABLE 10 Pharmacokinetic parameters of MI-219 following IVadministration at 2.0 mg/kg to Beagle dogs. T½ AUC0-t AUC0-∞ MRT Vss CLDog No. h ng · h/ml ng · h/ml h ml/kg ml/h/kg 1 4.32 8490 8512 1.22 286235 2 3.00 3884 3889 1.81 929 514 3 4.36 6272 6307 1.87 591 317 Mean3.90 6216 6236 1.63 602 355 SD 0.78 2303 2312 0.36 322 144 Min 3.00 38843889 1.22 286 235 Max 4.36 8490 8512 1.87 929 514 CV % 19.9 37.1 37.122.0 53.5 40.4

TABLE 11 Pharmacokinetic parameters of MI-219 following POadministration at 10 mg/kg to Beagle dogs. T½ Tmax Cmax AUC0-t AUC0-∞MRT F Dog No. h h ng/ml ng · h/ml ng · h/ml h % 1 2.77 1.0 3560 96659681 3.09 22.8 2 3.03 1.0 2119 5210 5226 2.71 26.8 3 2.81 0.5 3323 89678985 3.33 28.6 Mean 2.87 0.83 3001 7947 7964 3.04 26.1 SD 0.14 0.29 7732396 2396 0.31 3.0 Min 2.77 0.5 2119 5210 5226 2.71 22.8 Max 3.03 1.03560 9665 9681 3.33 28.6 CV % 4.9 35 25.8 30.2 30.1 10.2 11.5

TABLE 12 Plasma Concentration of MI-219 in Male Cynomolgus MonkeysFollowing Intravenous and Oral Administration. Sampling Time PointPlasma Concentration (ng/mL) IV (10 mg/kg) Animal Number 1 2 3 Mean SD0.083 15043.60 18669.25 16856.43 NA 0.1 18416.30 18416.30 NA 0.254144.21 4818.06 6877.15 5279.81 1423.78 0.5 2188.67 3577.55 3536.463100.89 790.28 1 1564.10 2277.17 2483.63 2108.30 482.46 1.5 1052.841867.36 2128.74 1682.98 561.15 2 985.23 1277.08 1539.44 1267.25 277.24 31230.40 691.83 834.00 918.74 279.11 4 348.97 582.63 562.45 498.02 129.476 246.12 211.39 324.99 260.83 58.21 8 180.91 115.94 202.16 166.34 44.9224 5.91 8.00 8.34 7.42 1.32 PO (50 mg/kg) Animal Number 4 5 6 Mean SD0.083 1.35 1.32 BLQ 1.33 NA 0.25 7.31 73.42 60.99 47.24 35.13 0.5 9.90107.48 165.45 94.28 78.61 1 192.53 186.66 241.02 206.74 29.83 1.5 521.16226.37 208.41 318.65 175.61 2 399.35 326.78 807.19 511.11 258.97 31497.78 691.63 859.30 1016.24 425.37 4 2219.11 526.86 618.28 1121.42951.73 6 1598.28 503.39 286.55 796.07 703.14 8 657.93 175.38 192.15341.82 273.89 24 24.18 18.74 9.89 17.60 7.21 BLQ: Below the limit ofquantitation; SD: Standard deviation; NA: Nonapplicable

TABLE 13 Selected Pharmacokinetics Parameters of MI-219 in MaleCynomolgus Monkeys Following Intravenous and Oral AdministrationPharmacokinetic Parameters Animal AUC_((0-t)) AUC_((0-∞)) MRT_((0-∞))T_(1/2) Vz CLz Cmax Tmax F No. h * μg/L h * μg/L h h L/kg L/h/kg μg/L h(%) IV 1 10271.39 10300.28 2.68 3.38 4.73 0.97 15043.60 2 11610.3011647.87 2.12 3.25 4.03 0.86 18669.25 3 13662.84 13703.99 2.44 3.43 3.620.73 18416.30 MEAN 11848.18 11884.05 2.41 3.35 4.12 0.85 17376.38 SD1708.19 1714.10 0.28 0.09 0.56 0.12 2024.20 Oral 1 14799.10 14905.546.25 3.03 NA NA 2219.11 4.00 25.08 2 4724.43 4732.32 6.38 2.52 NA NA691.63 3.00 7.96 3 5075.71 5128.70 5.57 3.72 NA NA 859.30 3.00 8.63 MEAN8199.74 8255.52 6.06 3.09 NA NA 1256.68 3.33 13.89 SD 5717.91 5762.500.44 0.60 NA NA 837.69 0.58 9.70 NA: Nonapplicable

Detailed studies were performed to determine the metabolism of MI-219 invitro using human and rat microsomes and in vivo using rats.

Metabolite Identification

Sample Preparation:

Metabolites in Liver Microsome Incubation:

MI-219 (or other compounds) was incubated with pooled liver microsomesin phosphate buffer at 37° C. The final concentrations of the compound,HLM, beta-NADPH, phosphate buffer and MgCl₂ were 20-50 μM, 1 mg/ml, 1mM, 0.1 M and 3.3 mM, respectively, in 0.4 ml of mixture solution. Thepercentage of MeOH in the incubation mixture was kept less than 0.2%(v/v). Samples were incubated for 60 min and the reaction was terminatedwith 1.2 mL of ice-cold acetonitrile to precipitate proteins. Twodifferent controls were prepared by using boiled microsomes (100° C. for5 min) or spiking MI-219 after protein precipitation. Samples weresubsequently centrifuged at 14,000 rpm for 5 min. The supernatant wasanalyzed by LC/MS/MS.

Metabolites in rat plasma: MI-219 was i.v. injected into maleSprague-Dawley rats (n=6, weight range 200-220 g) sat a dose of 5 mg/kg.MI-219 was i.v. injected at a dose of 5 mg/kg. Blood samples werecollected from the retro-orbital plexus of rats under light etheranesthesia into microfuge tubes containing heparin as an anti-coagulantat 0.166, 0.5, 1, 2, 4, 6, 8, and 24 h post-injection. Plasma washarvested by centrifuging the blood at 13000 rpm for 5 min at 4° C. andstored frozen at −80±10° C. until analysis.

Screening and characterization of metabolites with LC-MS/MS: MI-219 andother compounds were injected into mass spectrometer to obtain their MS,MS² and MS³ spectra. Based on the similarities and difference amongtheir mass spectra, the possible fragmentation pathways of protonatedMI-219 and several lead compounds were proposed. Three characteristicproduct ions were selected to generate 240 ion channels for MRMscreening by using Metabolite ID software (Applied Biosystems),including 40 common biotransformation processes. To search all themetabolites, two other scan modes, EMS full scan and precursor scan werealso conducted. Only the components detected in the sample and absent inall the control samples were regarded as possible metabolites. Toidentify the possible metabolites, both the sample and controls wereinjected on the LC-MS for EPI and MS³ scans to obtain their MS² and MS³spectra. Based on the MS², MS³ spectra of the metabolites and theproposed fragmentation pathways of MI-219 and other compounds, themetabolites were characterized.

FIG. 13A-D shows the MS/MS spectra of protonated MI-219, MI-142, MI-63and MI-708B. Based on these MS/MS spectra and MS³ spectra of the majorproduct ions such as m/z 496, 419, 363, 320, 285, 188, the fragmentationpathways of protonated MI-219 were proposed (Scheme 5). Supportingevidence for the proposed fragmentation was obtained by tracking thefunctional groups of the MI-219 derivatives. The presence and absence ofmass shifts of the product ions provide the structural information theproduct ions.

FIG. 14A-B shows the MS/MS spectra of deprotonated MI-219 and MI-142.The product ion m/z 306 was detected in the MS/MS spectra of both twocompounds. The product ion of MI-219 at m/z 243 and the product ion ofMI-142 at m/z 257 suggested the product ions of MI-219 at m/z 306 and243 were generated by cleaving the pyrrolidine ring. The metabolites ofMI-219 were tentatively elucidated by comparing their major product ionswith that of MI-219.

FIG. 15B shows the MS/MS spectrum of M1. The presence of m/z 419, 363,320 and 285 suggested that the metabolic site was not on the corestructure. The detection of product ions at m/z 186 and 104, which havea mass shift of 2 Da compared with m/z 188 and 106 of MI-219, suggestedthat the dehydrogenation occurred on the side chain. To confirm thestructure of M1, two ketone compounds were synthesized by oxidizingeither hydroxyl group of MI-219. The two compounds were found to beinterconvertible.

FIGS. 15A and 15C show that the synthesized compound exhibited the sameHPLC retention time and mass spectral pattern, suggesting that M1 isderived from the oxidation of one hydroxyl group of MI-219.

FIG. 16 shows the plasma concentrations of MI-219 and M1 in the rats.The elimination rates of the two compounds are similar.

Besides M1, another metabolite (M2) with a molecular weight of 567 Dawas detected at 4.76 min on the chromatogram. FIG. 17 shows the MS/MSspectra of protonated M2 (A) and deprotonated M2 (B). M2 showed a massshift of 16 Da compared with MI-219, suggesting that thebiotransformation was probably hydroxylation. The absence of m/z 419 and320 in FIG. 17A suggested that the hydroxylation occurred on the corestructure. The presence m/z 306 and 259 in FIG. 17B indicated that thehydroxylation occurred on the m/z 243 moiety of MI-219. Hence, it isinferred that the hydroxylation occurred on the3,3-dimethylbutan-1-amine moiety. The loss of 116 Da from m/z 568 to m/z452 is attributed to the elimination of hydroxylized3,3-dimethylbutan-1-amine radical. This radical loss of 116 Da was alsodetected in the hydroxylized metabolites of other MI-219 analogues suchas MI-773, MI-519-63 and MI-519-64, which is discussed later. Thehydroxylation of the amine is expected to facilitate the homolysis ofthe N—C bond. Hence, the hydroxyl group is assigned to the amine ofpyrrolidine ring.

Besides M1 and M2, the other 6 metabolites of MI-219 in human livermicrosome incubation were tentatively characterized. Their possiblestructures, chromatographic retention times, characteristic product ionsand peak areas are listed in Table 14.

TABLE 14 Metabolites of MI-219 in human liver microsome incubations [M +H]⁺ Peak Retention and area time product Metabolites (counts) (min) ions

5.7 × 10⁸ 6.52 550 [M + H]⁺ 419 363 320 235 186 104

8.1 × 10⁷ 4.76 568 [M + H]⁺ 452 347 319

7.7 × 10⁶ 1.1 × 10⁷ 6.19 and 6.28 566 [M + H]⁺ 492 419 320 210 182

2.4 × 10⁶ 6.77 522 [M + H]⁺ 419 320 235 220 158

4.1 × 10⁷ 6.55 532 [M + H]⁺ 419 320 220

2.4 × 10⁷ 6.20 568 [M + H]⁺ 464 419 320 204 122

8.9 × 10⁶ 1.5 × 10⁷ 5.9 × 10⁶ 6.34, 6.76, and 7.05 464 [M + H]⁺ 419 320285 153

3.2 × 10⁷ 6.57 632 [M + H]⁺ 550 533 419 320 225 168

Metabolism studies thus established that the primary metabolic site inMI-219 is on its “tail.” Furthermore, since two other promising MDM2inhibitors, MI-147 and MI-319, also contain the same diol tail, theirtail is also susceptible for quick metabolism. The compounds providedherein include new MDM2 inhibitors with different tails that may affordimproved metabolic stability. In addition, for the purpose of drugdevelopment, it is highly desirable to obtain new MDM2 inhibitors withnot only improved metabolic stability but also good oralbioavailability.

Direct modifications on the “diol” tail, which led to a series of newcompounds, e.g., MI-519-24, MI-519-28, MI-519-29, MI-519-31, and MI-758.Binding experiments showed that these new analogues bind to MDM2 withgood affinities (Table 19A). Furthermore, they also effectively inhibitcell growth in tumor cell lines with wild-type p53 and show selectivityover tumor cell lines with mutated or deleted p53, consistent with theirmechanism of action.

Microsomal stability studies showed that MI-758, MI-519-24, MI-519-28and MI-519-29 have an improved stability over MI-219 in rat livermicrosomes. MI-758 and MI-519-28 also have an improved stability overMI-219 in human liver microsomes. These data indicate that themicrosomal stabilities for the same compounds may be quite differentbetween rat and human microsomes (Table 15).

TABLE 15 Microsomal stability studies of MDM2 inhibitors. The microsomalstability of these compounds was evaluated together using the same batchof rat or human liver microsomes. Time (min) AT-219 MI-758 MI-519-24MI-519-28 MI-519-29 MI-519-31 Rat 0 100.00% 100.00% 100.00% 100.00%100.00% 100.00% Microsome 5 90.48% 96.52% 102.16% 92.34% 93.42% 80.22%10 80.58% 92.93% 86.33% 87.59% 84.91% 65.69% 15 72.12% 82.50% 75.72%86.13% 82.81% 57.57% 30 53.75% 73.32% 68.24% 71.97% 70.75% 42.91% 4548.85% 69.33% 66.08% 65.40% 71.89% 34.31% 60 49.62% 66.93% 61.51% 61.79%69.91% 32.81% T (½) min 33.15 79.82 48.52 74.56 61.30 18.69 Human 0100.00% 100.00% 100.00% 100.00% 100.00% 100.00% Microsome 5 97.51%95.88% 57.08% 91.87% 70.67% 78.11% 10 88.37% 89.21% 53.92% 89.02% 56.31%58.15% 15 81.51% 81.61% 49.62% 77.40% 49.52% 45.42% 30 58.25% 65.20%47.41% 66.83% 45.48% 22.75% 45 50.70% 54.52% 41.18% 55.85% 40.08% 13.25%60 42.15% 51.45% 41.42% 53.78% 35.28% 8.56% T (½) min 37.03 49.47 11.2454.11 12.07 13.01

Analogues containing different “tail” groups (Chart 2) were alsoinvestigated to further examine what kinds of tails would be more stablein rat or human liver microsomes. The microsomal stability data forthese analogues are summarized in Table 16. The microsomal stabilitydata showed that MI-122 and MI-126 have an improved rat microsomalstability over MI-219.

TABLE 16 Microsomal stability of MDM2 inhibitors in rat livermicrosomes. % of compound remaining When incubated in rat livermicrosomes at indicated time (min) 0 4 6 10 15 30 MI-219 (AT-219) 10076.6 64.3 57.3 51.9 49.3 MI-63 100 76 57.9 35.4 27.7 21 MI-120 100 95.188.1 84.7 82.1 79 MI-122 100 96.3 95.6 87.8 86.5 82.7 MI-130 100 77.260.1 48 42.3 34.1 MI-126 100 88.4 77.2 65.6 62.3 60.4 MI-129 100 74.452.5 41.1 36.7 33.1 MI-225 100 63.1 56.6 44.4 30.9 30.2

Based upon the improved microsomal stability, a series of new analogueswere designed and synthesized (Chart 3). The microsomal stability testshowed that MI-519-40, MI-519-43 and MI-763 have an improved microsomalstability over MI-219, whereas several others have comparable orinferior microsomal stability as compared to MI-219 (Table 17).

TABLE 17 Microsomal stability of previous synthesized MDM2 inhibitors inrat liver microsomes. % of compound remaining When incubated in ratliver microsomes at indicated time (min) 0 4 6 10 15 30 MI-219 (AT-219)100 76.6 64.3 57.3 51.9 49.3 MI-519-40 100 84.2 70.1 59.6 58.7 57.6MI-519-41 100 84.2 80.4 66.1 55.4 42.9 MI-519-43 100 92.3 86.1 70.6 69.962.5 MI-519-44 100 85.6 74.7 66.4 61.2 42.5 MI-519-45 100 81.1 59.6 50.744.3 38.5 MI-519-46 100 83.8 70.9 53.9 53.4 38.3 MI-519-47 100 80.1 61.145.3 38.1 34.3 MI-519-48 100 79.8 64.8 48.5 45.2 43.7 MI-748 100 79.169.7 61.5 49.9 43.7 MI-749 100 82.9 58.4 54.2 42.7 23 MI-751 100 83.162.9 50.1 41.6 31.1 MI-763 100 96.8 97.3 85.6 72.1 61.5 MI-764 100 87.876.5 66.5 63.9 55.8

Since it is highly desirable to obtain orally bioavailable compounds,several MDM2 inhibitors were evaluated for their pharmacokineticproperties in rats. The data are summarized in Table 18. Unfortunately,MI-122, MI-126, MI-519-24, MI-519-28 and MI-519-29, which have animproved microsomal stability over MI-219, have much inferior oralpharmacokinetic parameters in rats as compared to AT-219. One exceptionis MI-758, which shows good oral bioavailability and pharmacokineticparameters. These data indicate that it is difficult to design MDM2inhibitors with an improved microsomal stability and goodpharmacokinetic properties when dosing orally. Interestingly, MI-225,which has an inferior microsomal stability to MI-219, has a good oralbioavailability.

TABLE 18 Pharmacokinetic parameters of MDM2 inhibitors in male rats withoral dosing. Oral Dose C_(max) AUC_((0-t)) T_(1/2) Compounds (mg/kg)(ng/ml) (hr*mg/L) (hr) % F MI-219 25 3751 ± 1068 7677 ± 328  1.4 ± 0.165 MI-122 25 120 ± 112 1078 ± 891  7.1 ± 1.8 14 MI-126 25 274 ± 82  1888± 515  3.8 ± 0.5 31 MI-519-24 15 35 ± 23 101 ± 62  3.2 ± 1.4 1.1MI-519-28 15 102 ± 46  419 ± 239 1.6 ± 0.2 8.4 MI-519-29 15 51 ± 30 28 ±15 6.9 ± 7.5 <1 MI-758 15 1494 ± 1125 4282 ± 2690 1.9 ± 0.1 38 MI-225 251913 ± 312  7207 ± 1492 1.51 ± 0.12 44

Based upon these data, it is proposed that in order to achieve good oralbioavailability for MDM2 inhibitors, a charge neutral “tail” ispreferred. Furthermore, MI-225 achieved a good oral bioavailability,although it has an inferior microsomal stability to MI-219. Therefore, anumber of compounds containing a 4-, 5- and 6-membered ring with ahydroxyl group attached to it for the “tail” were prepared.

Biological testing showed that the configuration of the hydroxyl groupplays an important role for cellular activity, which was not expected.For example, MI-519-60 is more potent than its epimer MI-519-63, andMI-519-64 is more potent than its epimer MI-519-65, in inhibition ofcell growth in multiple cancer cell lines with wild-type p53 (Table19A).

Microsomal stability studies showed that while MI-519-51 has an inferiorstability to MI-219 in both human and rat liver microsomes, MI-773 ismore stable than MI-219 in human liver microsomes and MI-519-63 has acomparable microsomal stability to MI-219 (Tables 21-24).

Pharmacokinetic studies were performed on MI-519-51 and MI-773 and thedata are summarized in Tables 25-26. The PK data showed that whileMI-519-51 has inferior overall PK parameters to MI-219 in bothintravenous and oral routes of dosing, MI-773 has improved PK parametersover MI-219 in both intravenous and oral routes of dosing. For example,the AUC values in both routes of administration for MI-773 are 2-timeshigher than that for MI-219 at the same dose. These data indicated thatMI-773 has a good pharmacokinetic profile in both intravenous and oralroutes of dosing in rats and a good oral bioavailability. Furthermore,the major difference in their pharmacokinetic parameters betweenMI-519-51 and MI-773 cannot be predicted based upon their chemicalstructures.

To facilitate the design of new MDM2 inhibitors to further improve theiroverall PK profiles, metabolism studies on MI-773 and MI-519-63 wereperformed.

The major metabolites of MI-773 in human liver microsome incubation werecharacterized. FIGS. 18A-D show the MS/MS spectra of protonated MI-773and three metabolites of MI-773 (M1, M2 and M3). As shown in the MS/MSspectra of M1 and M2, the presence of m/z 419 and 320 suggested thedehydrogenation and hydroxylation did not occur on the core structure.The presence of m/z 114, a mass shift of 2 Da compared with m/z 116 ofprotonated MI-773, indicated that the metabolic sites located on thecyclohexane side chain. The chemical structures of MI-773 and M1 areshown in Chart 4.

The biotransformation pathways of MI-519-63 in human liver microsomeincubation were investigated. The detection of m/z 419, 363, 320 and 285in MS/MS spectra (FIGS. 19A-C) of both protonated MI-519-63 and M1indicated that the dehydrogenation occurred on the cyclobutane sidechain, which was further confirmed by the detection of the product ionof M1 at m/z 112, showing a mass shift of 2 Da compared with the production of MI-519-63 at m/z 114. The MS/MS spectrum of M2 showed a verysimilar pattern to that of MI-773-M3 and MI219-M2. The hydroxylation wasinferred to occur on the amine of pyrrolidine ring. The proposedbiotransformations of MI-519-63 are shown in Chart 5.

Compounds provided herein include spiro-oxindole MDM2 inhibitors thatwere designed based upon these metabolism studies of MI-773 andMI-519-63.

Binding and cellular studies showed that while MI-519-64 and MI-519-65bind to human MDM2 protein with high affinities and effectively inhibitcancer cell growth in cancer cell lines with wild-type p53, MI-519-64 isseveral times more potent than MI-519-65.

Microsomal stability studies showed that MI-519-64 has an improvedmicrosomal stability over MI-519-63 (Tables 22-24).

Since the hydroxyl group in MI-773 and MI-519-63 can be metabolized togive a ketone, replacement of this hydroxyl with a sulphonylamide groupwas investigated as a means to generate MDM2 inhibitors with improvedmicrosomal stability and/or good oral bioavailability (see MI-771 andMI-772).

Binding and cellular studies showed that while both MI-771 and MI-772bind to human MDM2 protein with high affinities and effectively inhibitcell growth in cancer cell lines with wild-type p53, MI-772 is severaltimes more potent than MI-771.

Microsomal stability studies showed that MI-772 is very stable, muchmore stable than MI-773 and also MI-219 (Tables 21 and 23).Surprisingly, MI-771 has a very poor microsomal stability (Tables 21 and23). The microsomal stability data on MI-772 and its epimer MI-771 showthat the configuration of the —NHSO₂Me group has a substantial impact onthe microsomal stability of these compounds, which was not expected.

Pharmacokinetic studies in rats showed that MI-772 has a long half-lifein rats in both intravenous and oral routes of dosing (Tables 25 and 26)and has a modest oral bioavailability. In comparison, MI-771 has a verypoor pharmacokinetic profile in both intravenous and oral routes ofdosing. Thus, the configuration of the —NHSO₂Me group in MI-771 andMI-772 has a substantial impact on their pharmacokinetic profiles.

Example 1 Analytical Data for Compounds MI-219-M1(b)—TFA Salt

¹H NMR (300 MHz, CD₃OD) δ 7.72 (d, J=8.5 Hz, 1H), 7.32-7.21 (m, 3H),7.08 (d, J=7.7 Hz, 1H), 6.87 (d, J=6.0 Hz, 1H), 5.25 (d, J=11.2 Hz, 1H),4.50-4.47 (m, 1H), 4.11 (d, J=11.2 Hz, 1H), 4.05-3.97 (m, 2H), 3.67-3.38(m, 2H), 2.61-2.56 (m, 2H), 1.92 (dd, J=15.5, 8.2 Hz, 1H), 1.20 (dd,J=15.5, 2.0 Hz, 1H), 0.92 (s, 9H); MS (ESI) m/z 550 [M+H]⁺.

MI-519-24—TFA Salt

¹H NMR (300 MHz, CD₃OD) δ 7.70 (m, 1H), 7.31-7.02 (m, 4H), 6.88-6.83 (m,1H), 5.28 (d, J=11.3 Hz, 1H), 4.48-4.45 (m, 1H), 4.18-4.13 (m, 1H),3.75-3.72 (m, 2H), 3.36-3.31 (m, 1H), 3.05-3.03 (m, 1H), 2.83-2.76 (m,1H), 1.92 (dd, J=15.4, 8.4 Hz, 1H), 1.65-1.53 (2H), 1.36 (s, 9H), 1.17(d, J=15.4 Hz, 1H), 0.92 (s, 9H); MS (ESI) m/z 607 [M+H]⁺.

MI-519-27—TFA Salt

¹H NMR (300 MHz, CD₃OD) δ 7.68 (d, J=8.5 Hz, 1H), 7.32-7.20 (m, 3H),7.08 (d, J=7.7 Hz, 1H), 6.87 (d, J=6.0 Hz, 1H), 5.20 (d, J=11.2 Hz, 1H),4.38 (d, J=7.8 Hz, 1H), 4.24-4.20 (m, 2H), 4.08 (d, J=11.2 Hz, 1H),2.19-2.12 (m, 1H), 1.90-1.70 (m, 3H), 1.53-1.48 (m, 2H), 1.39-1.31 (m,1H), 1.20-1.16 (m, 1H), 0.91 (s, 9H); MS (ESI) m/z 548 [M+H]⁺.

MI-519-29—TFA Salt

¹H NMR (300 MHz, CD₃OD) δ 7.70 (d, J=8.5 Hz, 1H), 7.33-7.21 (m, 3H),7.09 (d, J=7.6 Hz, 1H), 6.87 (d, J=6.0 Hz, 1H), 5.27 (d, J=11.2 Hz, 1H),4.56 (s, 1H), 4.46-4.44 (m, 1H), 4.15 (d, J=11.2 Hz, 1H), 3.75-3.65(4H), 3.42-3.28 (m, 2H), 3.15 (m, 3H), 2.30-2.05 (m, 2H), 1.90 (dd,J=15.4, 8.5 Hz, 1H).

MI-519-30—TFA Salt

¹H NMR (300 MHz, CD₃OD) δ 7.55 (d, J=8.7 Hz, 1H), 7.23-7.14 (m, 2H),7.08 (s, 1H), 7.02-6.99 (m, 1H), 6.85 (d, J=6.2 Hz, 1H), 4.87 (d, J=11.0Hz, 1H), 4.27-4.24 (m, 1H), 4.11-4.06 (m, 1H), 3.50-3.47 (m, 1H),3.40-3.32 (m, 2H), 3.19-3.16 (m, 1H), 1.58-1.48 (m, 3H), 1.12-1.04 (m,4H), 0.92 (s, 9H); MS (ESI) m/z 566 [M+H]⁺.

MI-519-35—TFA Salt

¹H NMR (300 MHz, CD₃OD) δ 7.68 (d, J=8.5 Hz, 1H), 7.31-7.20 (m, 3H),7.10-7.08 (m, 1H), 6.86 (d, J=6.0 Hz, 1H), 5.26 (d, J=11.2 Hz, 1H),5.10-5.03 (m, 1H), 4.50 (m, 1H), 4.40-4.37 (m, 1H), 4.16 (d, J=11.2 Hz,1H), 3.63-3.51 (m, 4H), 3.44-3.32 (m, 3H), 2.21-2.18 (m, 1H), 2.01-1.95(m, 1H), 1.89 (dd, J=15.4, 8.6 Hz, 1H), 1.18-1.03 (m, 1H), 0.92 (s, 9H);MS (ESI) m/z 577 [M+H]⁺.

MI-519-36—TFA Salt

¹H NMR (300 MHz, CD₃OD) δ 7.53 (m, 1H), 7.27-7.17 (m, 2H), 7.07 (s, 1H),7.00-6.98 (m, 1H), 6.86 (d, J=6.2 Hz, 1H), 4.98 (d, J=12.4 Hz, 1H),4.45-4.41 (m, 1H), 4.10 (d, J=12.4 Hz, 1H), 3.85-3.81 (m, 1H), 3.28-3.22(m, 1H), 3.04-2.96 (m, 1H), 2.33-2.29 (m, 1H), 1.74 (dd, J=15.2, 7.3 Hz,1H), 1.64-1.51 (m, 2H), 1.30-1.21 (m, 3H), 0.90 (s, 9H); MS (ESI) m/z578 [M+H]⁺.

MI-519-37—TFA Salt

¹H NMR (300 MHz, CD₃OD) δ 7.71 (d, J=8.5 Hz, 1H), 7.33-7.22 (m, 3H),7.08 (d, J=7.6 Hz, 1H), 6.87 (d, J=6.0 Hz, 1H), 5.26 (d, J=11.3 Hz, 1H),4.49-4.46 (m, 1H), 4.10 (d, J=11.3 Hz, 1H), 3.85-3.78 (m, 2H), 3.33-3.22(m, 1H), 3.00-2.93 (m, 1H), 2.31-2.28 (m, 1H), 1.92 (dd, J=15.5, 8.2 Hz,1H), 1.60-1.52 (m, 2H), 1.28-1.18 (m, 3H), 0.92 (s, 9H); MS (ESI) m/z578 [M+H]⁺.

MI-519-41—TFA Salt

¹H NMR (300 MHz, CD₃OD) δ 7.68 (d, J=8.5 Hz, 1H), 7.33-7.24 (m, 3H),7.10 (d, J=7.7 Hz, 1H), 6.87 (d, J=6.0 Hz, 1H), 5.28 (d, J=11.2 Hz, 1H),4.44 (m, 1H), 4.11 (d, J=11.2 Hz, 1H), 1.90 (dd, J=15.5, 8.3 Hz, 1H),1.20 (dd, J=15.5, 2.0 Hz, 1H), 0.92 (s, 9H); MS (ESI) m/z 464 [M+H]⁺.

MI-519-43—TFA Salt

¹H NMR (300 MHz, CD₃OD) δ 7.57-7.54 (m, 2H), 7.46-7.40 (m, 1H),7.22-7.14 (m, 2H), 6.81 (d, J=1.8 Hz, 1H), 5.34 (d, J=11.4 Hz, 1H), 4.66(d, J=11.4 Hz, 1H), 4.55-4.52 (m, 1H), 3.60 (m, 2H), 3.32-3.26 (m, 2H),3.03-2.93 (m, 4H), 2.11-1.81 (m, 7H), 1.15 (dd, J=15.4, 1.7 Hz, 1H),0.90 (s, 9H); MS (ESI) m/z 575 [M+H]⁺.

MI-519-44—TFA Salt

¹H NMR (300 MHz, CD₃OD) δ 7.63-7.57 (m, 2H), 7.46-7.41 (m, 1H),7.23-7.14 (m, 2H), 6.81 (m, 1H), 5.30 (d, J=11.3 Hz, 1H), 4.64 (d,J=11.3 Hz, 1H), 4.50 (d, J=8.3 Hz, 1H), 3.43-3.19 (m, 4H), 2.94-2.75 (m,4H), 1.95-1.71 (m, 8H), 1.53-1.43 (m, 1H), 1.14 (d, J=15.2 Hz, 1H), 0.92(s, 9H); MS (ESI) m/z 589 [M+H]⁺.

MI-519-49—TFA Salt

¹H NMR (300 MHz, CD₃OD) δ 7.60-7.55 (m, 2H), 7.44-7.39 (m, 1H),7.20-7.15 (m, 2H), 6.80 (d, J=1.8 Hz, 1H), 5.33 (d, J=11.4 Hz, 1H), 4.64(d, J=11.4 Hz, 1H), 4.48 (d, J=7.1 Hz, 1H), 3.72-3.64 (m, 1H), 3.55-3.52(m, 5H), 3.38-3.30 (m, 6H), 2.95 (s, 3H), 1.93 (dd, J=15.3, 8.6 Hz, 1H),1.13 (d, J=15.3 Hz, 1H), 0.90 (s, 9H); MS (ESI) m/z 654 [M+H]⁺.

MI-519-50—TFA Salt

¹H NMR (300 MHz, CD₃OD) δ 7.58-7.55 (m, 2H), 7.45-7.39 (m, 1H),7.22-7.14 (m, 2H), 6.80 (d, J=1.8 Hz, 1H), 5.29 (d, J=11.3 Hz, 1H), 4.60(d, J=11.3 Hz, 1H), 4.50 (dd, J=8.6, 1.9 Hz, 1H), 1.88 (dd, J=15.4, 8.4Hz, 1H), 1.15 (dd, J=15.4, 1.9 Hz, 1H), 0.90 (s, 9H); MS (ESI) m/z 464[M+H]⁺.

MI-519-51—TFA Salt

¹H NMR (300 MHz, CD₃OD) δ 7.54 (d, J=8.1 Hz, 1H), 7.36-7.31 (m, 1H),7.15-7.12 (m, 1H), 7.02-6.91 (m, 2H), 6.81-6.80 (m, 1H), 4.98 (d, J=11.9Hz, 1H), 4.47 (d, J=11.8 Hz, 1H), 4.41-4.37 (m, 1H), 4.22-4.19 (m, 2H),2.18-2.08 (m, 1H), 1.85-1.49 (m, 5H), 1.37-1.33 (m, 1H), 1.14 (dd,J=15.2, 3.6 Hz, 1H), 0.91 (s, 9H); MS (ESI) m/z 548 [M+H]⁺.

MI-519-51-epi—TFA Salt

¹H NMR (300 MHz, CD₃OD) δ 7.63-7.56 (m, 2H), 7.45-7.40 (m, 1H),7.21-7.14 (m, 2H), 6.82 (m, 1H), 5.19 (d, J=11.3 Hz, 1H), 4.59 (d,J=11.3 Hz, 1H), 4.54-4.51 (m, 1H), 4.35-4.32 (m, 1H), 4.24-4.21 (m, 1H),2.02-1.85 (m, 3H), 1.73-1.63 (m, 2H), 1.51 (m, 1H), 1.19-1.14 (m, 1H),1.09-1.04 (m, 1H), 0.90 (s, 9H); MS (ESI) m/z 548 [M+H]⁺.

MI-519-60—TFA Salt

¹H NMR (300 MHz, CD₃OD) δ 7.61-7.56 (m, 1H), 7.41-7.36 (m, 1H),7.23-7.18 (m, 1H), 6.88 (m, 1H), 6.80-6.76 (m, 1H), 6.66-6.60 (m, 1H),4.82-4.77 (m, 1H), 4.43-4.15 (m, 2H), 4.05-3.80 (m, 2H), 3.75-3.51 (m,3H), 2.35-2.09 (m, 1H), 1.88 (dd, J=15.0, 8.0 Hz, 1H), 1.24-1.18 (m,1H), 0.82 (s, 9H); MS (ESI) m/z 534 [M+H]⁺.

MI-519-56—TFA Salt

¹H NMR (300 MHz, CD₃OD) δ 7.63-7.57 (m, 2H), 7.44-7.39 (m, 1H),7.21-7.15 (m, 2H), 6.81 (m, 1H), 5.26 (d, J=10.9 Hz, 1H), 4.75-4.73 (m,1H), 4.64 (d, J=11.4 Hz, 1H), 4.52-4.50 (m, 1H), 3.98-3.94 (m, 1H),3.75-3.60 (m, 1H), 3.55-3.52 (m, 1H), 3.46-3.42 (m, 1H), 3.31-3.13 (m,1H), 2.31-2.20 (m, 1H), 1.95-1.87 (m, 3H), 1.80-1.50 (m, 1H), 1.18-1.13(m, 1H), 0.90 (s, 9H); MS (ESI) m/z 593 [M+H]⁺.

MI-519-62—TFA Salt

¹H NMR (300 MHz, CD₃OD) δ 7.57-7.53 (m, 1H), 7.41-7.38 (m, 1H),7.22-7.17 (m, 1H), 6.87-6.82 (m, 3H), 5.02 (d, J=10.1 Hz, 1H), 4.44 (d,J=10.1 Hz, 1H), 4.11 (dd, J=7.4, 2.8 Hz, 1H), 3.41-3.36 (m, 1H),3.12-3.07 (m, 1H), 1.97 (dd, J=15.2, 7.5 Hz, 1H), 1.32 (dd, J=15.2, 2.9Hz, 1H), 1.07 (s, 3H), 0.98 (s, 3H), 0.90 (s, 9H); MS (ESI) m/z 536[M+H]⁺.

MI-519-63—TFA Salt

¹H NMR (300 MHz, CD₃OD) δ 7.55-7.51 (m, 1H), 7.43-7.38 (m, 1H),7.24-7.18 (m, 1H), 6.88 (m, 1H), 6.81 (m, 1H), 6.79-6.73 (m, 1H), 4.93(d, J=9.7 Hz, 1H), 4.40 (d, J=9.7 Hz, 1H), 4.11 (dd, J=7.7, 2.7 Hz, 1H),3.98-3.93 (m, 1H), 3.87-3.32 (m, 1H), 2.72-2.56 (m, 2H), 2.03-1.87 (m,2H), 1.77-1.71 (m, 1H), 1.25 (dd, J=15.4, 2.6 Hz, 1H), 0.89 (s, 9H); MS(ESI) m/z 534 [M+H]⁺.

MI-519-68—TFA Salt

¹H NMR (300 MHz, CD₃OD) δ 7.58-7.55 (m, 1H), 7.38-7.35 (m, 1H),7.21-7.16 (m, 1H), 6.87 (s, 1H), 6.82-6.78 (m, 2H), 5.08 (d, J=10.0 Hz,1H), 4.45 (d, J=10.0 Hz, 1H), 4.17-4.07 (m, 2H), 3.78 (s, 2H), 2.68-2.58(m, 2H), 2.44-2.40 (m, 1H), 2.30-2.27 (m, 1H), 1.98 (dd, J=15.4, 7.7 Hz,1H), 1.58 (s, 1H), 1.26 (dd, J=15.4, 2.5 Hz, 1H), 0.82 (s, 9H); MS (ESI)m/z 464 [M+H]⁺.

MI-519-69—TFA Salt

¹H NMR (300 MHz, CD₃OD) δ 7.62-7.57 (m, 1H), 7.41-7.35 (m, 1H),7.22-7.17 (m, 1H), 6.88 (s, 1H), 6.79 (m, 2H), 5.11 (d, J=10.1 Hz, 1H),4.47 (d, J=10.1 Hz, 1H), 4.19 (dd, J=7.6, 2.6 Hz, 1H), 4.07-4.02 (m,1H), 3.82 (s, 2H), 2.87-2.74 (m, 2H), 2.22-2.18 (m, 1H), 2.07-1.96 (m,2H), 1.27 (dd, J=15.3, 2.6 Hz, 1H), 0.82 (s, 9H); MS (ESI) m/z 591[M+H]⁺.

MI-519-70—TFA Salt

¹H NMR (300 MHz, CD₃OD) δ 7.58-7.53 (m, 1H), 7.44-7.39 (m, 1H),7.26-7.21 (m, 1H), 6.90-6.89 (m, 1H), 6.80-6.77 (m, 1H), 6.60 (d, J=8.1Hz, 1H), 5.00 (d, J=9.0 Hz, 1H), 4.50-4.40 (m, 2H), 4.08 (d, J=5.5 Hz,1H), 3.43-3.32 (m, 2H), 3.17-3.06 (m, 2H), 1.96 (dd, J=15.2, 7.9 Hz,1H), 1.18 (dd, J=15.2, 1.9 Hz, 1H), 0.83 (s, 9H); MS (ESI) m/z 532[M+H]⁺.

¹H NMR (300 MHz, CD₃OD) δ 7.65 (d, J=8.4 Hz, 1H), 7.29 (d, J=7.8 Hz,1H), 7.27 (s, 1H), 7.21 (t, J=7.8 Hz, 1H), 7.04 (d, J=7.5 Hz, 1H), 6.83(d, J=6.0 Hz, 1H), 5.27 (d, J=11.4 Hz, 1H), 4.41 (d, J=6.6 Hz, 1H), 4.10(d, J=11.4 Hz, 1H), 3.39-3.53 (m, 1H), 3.11-3.22 (m, 1H), 3.00 (s, 4H),2.53 (s, 4H), 2.41 (dd, J=6.6, 12.0 Hz, 1H), 1.86 (dd, J=8.1, 15.3 Hz,1H), 1.09-1.38 (m, 2H), 0.93 (s, 9H).

¹H NMR (300 MHz, CD₃OD) δ 7.66 (d, J=8.4 Hz, 1H), 7.15-7.33 (m, 3H),7.05 (d, J=7.5 Hz, 1H), 6.81 (d, J=6.0 Hz, 1H), 5.31 (d, J=11.4 Hz, 1H),4.44 (dd, J=1.8, 8.4 Hz, 1H), 4.12 (d, J=11.4 Hz, 1H), 3.33-3.45 (m,1H), 3.18 (dd, J=7.5, 14.7 Hz, 4H), 2.95-3.10 (m, 1H), 2.80 (s, 3H),2.66 (dd, J=7.5, 13.8 Hz, 4H), 2.46 (dd, J=6.6, 12.6 Hz, 2H), 1.88 (dd,J=8.4, 15.3 Hz, 1H), 1.23-1.38 (m, 1H), 0.85 (s, 9H).

¹H NMR (300 MHz, CD₃OD) δ 7.68 (d, J=8.4 Hz, 1H), 7.13-7.30 (m, 3H),7.04 (d, J=7.2 Hz, 1H), 6.84 (d, J=6.0 Hz, 1H), 5.32 (d, J=11.4 Hz, 1H),4.41 (dd, J=1.8, 8.4 Hz, 1H), 4.11 (d, J=11.4 Hz, 1H), 3.31-3.46 (m,1H), 3.16 (dd, J=7.5, 14.7 Hz, 4H), 2.95-3.10 (m, 1H), 2.78 (s, 3H),2.67 (dd, J=7.5, 14.1 Hz, 4H), 2.46 (dd, J=6.6, 12.6 Hz, 2H), 2.09-2.38(m, 2H), 1.67-1.78 (m, 1H), 1.23-1.38 (m, 1H), 0.86 (s, 9H).

¹H NMR (300 MHz, CD₃OD) δ 7.70 (d, J=8.4 Hz, 1H), 7.58 (t, J=7.2 Hz,1H), 7.42 (t, J=7.5 Hz, 1H), 7.18 (t, J=8.1 Hz, 1H), 6.88 (d, J=6.0 Hz,1H), 5.31 (d, J=11.4 Hz, 1H), 4.67 (d, J=11.4 Hz, 1H), 4.48 (d, J=7.8Hz, 1H), 3.52-3.80 (m, 2H), 3.00-3.48 (m, 6H), 1.95-2.30 (m, 4H), 1.93(dd, J=8.4, 15.3 Hz, 1H), 1.13 (d, J=15.6 Hz, 1H), 0.92 (s, 9H).

¹H NMR (300 MHz, CD₃OD) δ 7.71 (d, J=8.1 Hz, 1H), 7.60 (t, J=7.2 Hz,1H), 7.45 (t, J=6.9 Hz, 1H), 7.21 (t, J=7.8 Hz, 1H), 6.89 (d, J=6.0 Hz,1H), 5.30 (d, J=11.4 Hz, 1H), 4.66 (d, J=11.4 Hz, 1H), 4.52 (d, J=8.1Hz, 1H), 3.55-3.80 (m, 2H), 3.18-3.46 (m, 2H), 2.82-3.15 (m, 4H),1.74-2.33 (m, 7H), 1.16 (d, J=15.6 Hz, 1H), 0.92 (s, 9H).

¹H NMR (300 MHz, CD₃OD) δ 7.71 (d, J=8.7 Hz, 1H), 7.59 (t, J=6.6 Hz,1H), 7.44 (t, J=6.9 Hz, 1H), 7.20 (t, J=8.1 Hz, 1H), 6.88 (d, J=6.0 Hz,1H), 5.28 (d, J=11.4 Hz, 1H), 4.65 (d, J=11.4 Hz, 1H), 4.52 (d, J=8.4Hz, 1H), 3.55-3.70 (m, 2H), 3.08-3.27 (m, 4H), 2.88-3.08 (m, 2H),1.98-2.28 (m, 5H), 1.91 (dd, J=8.4, 15.3 Hz, 1H), 1.40-1.65 (m, 4H),1.15 (d, J=15.3 Hz, 1H), 0.91 (s, 9H), 0.82 (d, J=9.9 Hz, 1H).

¹H NMR (300 MHz, CD₃OD) δ 8.33 (d, J=7.5 Hz, 1H), 7.45 (t, J=6.9 Hz,1H), 7.34 (t, J=7.8 Hz, 1H), 7.15 (t, J=8.7 Hz, 1H), 6.83 (d, J=1.5 Hz,1H), 6.75 (dd, J=1.8, 8.1 Hz, 1H), 6.66 (d, J=8.1 Hz, 1H), 4.79 (d,J=9.9 Hz, 1H), 4.32 (d, J=9.6 Hz, 1H), 3.99 (d, J=5.4 Hz, 1H), 3.55-3.70(m, 1H), 3.05-3.20 (m, 1H), 2.89 (s, 3H), 1.78-2.06 (m, 4H), 1.67 (d,J=12.6 Hz, 1H), 1.15-1.45 (m, 6H), 0.86 (d, J=6.3 Hz, 1H), 0.78 (s, 9H).

¹H NMR (300 MHz, CD₃OD) δ 8.31 (d, J=7.8 Hz, 1H), 7.46 (t, J=6.9 Hz,1H), 7.34 (t, J=7.8 Hz, 1H), 7.15 (t, J=8.7 Hz, 1H), 6.83 (d, J=1.5 Hz,1H), 6.75 (dd, J=1.8, 8.1 Hz, 1H), 6.66 (d, J=8.1 Hz, 1H), 4.79 (d,J=9.9 Hz, 1H), 4.32 (d, J=9.6 Hz, 1H), 3.99 (d, J=5.4 Hz, 1H), 3.55-3.70(m, 1H), 3.05-3.20 (m, 1H), 2.89 (s, 3H), 1.78-2.06 (m, 4H), 1.67 (d,J=12.6 Hz, 1H), 1.15-1.45 (m, 6H), 0.86 (d, J=6.3 Hz, 1H), 0.78 (s, 9H).

¹H NMR (300 MHz, CD₃OD) δ 7.48 (t, J=6.6 Hz, 1H), 7.35 (t, J=6.9 Hz,1H), 7.16 (t, J=7.8 Hz, 1H), 6.83 (d, J=1.8 Hz, 1H), 6.75 (dd, J=1.8,8.1 Hz, 1H), 6.65 (d, J=8.1 Hz, 1H), 4.33 (d, J=9.3 Hz, 1H), 4.02 (d,J=5.4 Hz, 1H), 3.70-3.88 (m, 1H), 3.62 (d, J=12.3 Hz, 1H), 3.53 (d,J=12.9 Hz, 1H), 2.77 (s, 3H), 2.65-2.90 (m, 2H), 1.79-2.06 (m, 2H),1.65-1.78 (m, 1H), 1.47-1.63 (m, 1H), 1.32-1.47 (m, 1H), 1.10-1.24 (m,1H), 0.86 (d, J=6.0 Hz, 1H), 0.78 (s, 9H), 0.76 (d, J=11.7 Hz, 1H).

¹H NMR (300 MHz, CD₃OD) δ 8.53 (d, J=7.5 Hz, 1H), 7.55 (t, J=7.5 Hz,1H), 7.39 (t, J=7.2 Hz, 1H), 7.19 (t, J=8.1 Hz, 1H), 6.88 (s, 1H), 6.82(s, 2H), 5.04 (d, J=9.9 Hz, 1H), 4.46 (d, J=9.9 Hz, 1H), 4.20 (dd,J=2.7, 7.2 Hz, 1H), 3.73-4.05 (m, 2H), 3.07-3.22 (m, 2H), 2.86 (dd,J=13.8, 26.1 Hz, 2H), 2.03 (dd, J=7.2, 15.3 Hz, 1H), 1.78-1.96 (m, 2H),1.56-1.68 (m, 1H), 1.16-1.53 (m, 3H), 1.10 (t, J=7.2 Hz, 3H), 0.90 (d,J=4.5 Hz, 1H), 0.82 (s, 9H), 0.80 (d, J=12.9 Hz, 1H).

C027—TFA Salt

¹H NMR (300 MHz, MeOH-d4): 7.50-7.36 (m, 1H), 7.24-7.10 (m, 2H),6.88-6.76 (m, 3H), 5.12 (d, J=10.17 Hz, 1H), 4.49 (d, J=10.17 Hz, 1H),4.23 (dd, J=6.83, 2.09 Hz, 1H), 3.98-3.83 (m, 1H), 2.49-2.36 (m, 1H),2.36-2.22 (m, 1H), 2.10-1.96 (m, 2H), 1.94-1.82 (m, 1H), 1.35-1.28 (m,1H), 1.29 (s, 3H), 0.80 (s, 9H); ¹³C NMR (75 MHz, MeOH-d4): 108.1,166.0, 145.4, 136.9, 127.9, 126.1 (t, J_(C-F)=5.6 Hz), 125.4, 123.4118.8 (d, J_(C-F)=17.3 Hz), 112.0, 67.4, 64.5, 63.7, 61.6, 49.5, 45.6,45.5, 42.4, 38.5, 30.9, 29.5, 27.6; ESI-MS calculated for C₂₈H₃₃³⁵ClF₂N₃O₃[M+H]⁺: 532.2179. Found: 532.42.

C029—TFA Salt

¹H NMR (300 MHz, MeOH-d4): 8.84 (d, J=6.80 Hz, 1H), 7.58 (t, J=6.80 Hz,1H), 7.39 (t, J=7.11 Hz, 1H), 7.22 (t, J=7.80 Hz, 1H), 6.88 (dd, J=9.81,7.80 Hz, 1H), 6.78 (d, J=10.13, 6.63 Hz, 1H), 5.11 (d, J=10.37 Hz, 1H),4.48 (d, J=10.37 Hz, 1H), 4.21 (d, J=10.37 Hz, 1H), 4.21 (dd, J=7.32,2.66 Hz, 1H), 3.95-3.75 (m, 1H), 2.46-2.22 (m, 2H), 2.12-1.96 (m, 2H),1.94-1.80 (m, 1H), 1.34-1.28 (m, 1), 1.29 (s, 3H), 0.81 (s, 9H); ¹³C NMR(75 MHz, MeOH-d4): 180.2, 169.2, 132.2, 128.7 (d, J_(C-F)=2.2 Hz), 126.5(d, J_(C-F)=4.6 Hz), 124.7 (dd, J_(C-F)=33.5, 19.2 Hz), 122.6 (d,J_(C-F)=18.1 Hz), 101.5 (d, J_(C-F)=23.0 Hz), 67.4, 64.4, 63.5, 61.9,49.8, 45.6, 45.5, 42.4, 38.6, 30.9, 29.5, 27.6; ESI-MS calculated forC₂₈H₃₂ ³⁵ClF₃N₃O₃ [M+H]⁺: 550.2084. Found: 550.33.

C031—TFA Salt

¹H NMR (300 MHz, MeOH-d4): 7.68-7.54 (m, 1H), 7.38-7.26 (m, 1H),7.22-7.12 (m, 1H), 6.90-6.76 (m, 1H), 6.70-6.60 (m, 1H), 6.56-6.42 (m,1H), 5.30-5.20 (m, 1H), 4.49 (d, J=10.03 Hz, 1H), 4.25 (dd, J=71.9, 2.39Hz, 1H), 4.00-3.82 (m, 1H), 2.50-2.21 (m, 2H), 2.18-2.00 (m, 2H),1.98-1.82 (m, 1H), 1.40-1.30 (m, 1H), 1.28 (s, 3H), 0.79 (s, 9H); ¹³CNMR (75 MHz, MeOH-d4): 180.6, 165.1 (d, J_(C-F)=246.7 Hz), 166.1, 157.7(d, J_(C-F)=247.9 Hz), 145.6 (d, J_(C-F)=12.0 Hz), 132.0, 128.6, 128.2(d, J_(C-F)=10.2 Hz), 126.3 (d, J_(C-F)=4.5 Hz), 125.0 (d, J_(C-F)=14.0Hz), 122.4 (d, J_(C-F)=18.4 Hz), 122.3, 109.8 (d, J_(C-F)=23.2 Hz), 99.9(d, J_(C-F)=27.8 Hz), 67.4, 64.5, 63.5, 61.5, 49.2, 45.6, 45.5, 42.3,38.4, 30.9, 29.5, 27.5; ESI-MS calculated for C₂₈H₃₃ ³⁵ClF₂N₃O₃ [M+H]⁺:532.2179. Found: 532.42.

C034—TFA Salt

¹H NMR (300 MHz, MeOH-d4): 7.28-7.10 (m, 5H), 6.92-6.84 (m, 1H),6.80-6.76 (m, 1H), 5.40-5.20 (m, 1H), 5.08 (d, J=10.96 Hz, 1H),4.40-4.20 (m, 1H), 3.90-3.60 (m, 1H), 2.50-2.30 (m, 1H), 2.30-2.15 (m,1H), 2.15-2.00 (m, 2H), 1.90-1.75 (m, 1H), 1.57 (dd, J=15.3, 3.71 Hz,1H), 1.25 (s, 3H), 0.79 (s, 9H); ¹³C NMR (75 MHz, MeOH-d4): 180.0,165.9, 144.7, 136.7, 136.6, 135.8, 131.3, 130.1, 129.8, 128.1, 128.1,126.8, 123.5, 112.0, 67.4, 64.3, 64.0, 62.2, 57.2, 45.7, 45.6, 42.7,38.3, 31.0, 29.6, 27.5; ESI-MS calculated for C₂₈H₃₄ ³⁵Cl₂N₃O₃ [M+H]⁺:530.1977. Found: 530.50.

C035—TFA Salt

¹H NMR (300 MHz, MeOH-d4): 7.40-7.00 (m, 5H), 6.80-6.40 (m, 1H),5.60-5.00 (m, 2H), 4.60-4.20 (m, 1H), 4.00-3.80 (m, 1H), 2.60-2.40 (m,1H), 2.40-2.20 (m, 1H), 2.20-2.00 (m, 2H), 2.00-1.80 (m, 1H), 1.70-1.50(m, 1H), 1.28 (s, 3H), 0.83 (s, 9H); ¹³C NMR (75 MHz, MeOH-d4): 180.0,165.8, 160.0-145.0 (m, 2×C_(sp2)-F), 136.5, 135.9, 131.4, 130.0, 129.9,128.0, 124.1 (d, J_(C-F)=6.3 Hz), 119.1, 116.7 (d, J_(C-F)=20.4 Hz),101.4 (d, J_(C-F)=23.0 Hz), 67.4, 64.2, 63.8, 62.5, 57.4, 45.6, 45.5,42.7, 38.3, 31.0, 29.5, 27.5; ESI-MS calculated for C₂₈H₃₃³⁵ClF₂N₃O₃[M+H]⁺: 532.2179. Found: 532.42.

MI-519-73—TFA Salt

¹H NMR (300 MHz, MeOH-d4): 7.50-7.30 (m, 2H), 7.20-7.10 (m, 1H),6.90-6.70 (m, 3H), 5.00-4.70 (m, 1H), 4.36 (d, J=9.76 Hz, 1H), 4.05-3.96(m, 1H), 3.70-3.50 (m, 1H), 1.94 (dd, J=14.98, 7.30 Hz, 1H), 1.80-1.00(m, 8H), 1.16 (s, 3H), 0.90-0.70 (m, 1H), 0.80 (s, 9H); ESI-MScalculated for C₃₀H₃₇ ³⁵Cl₂FN₃O₃ [M+H]⁺: 576.2196. Found: 576.58.

MI-519-74—TFA salt

¹H NMR (300 MHz, MeOH-d4): 7.50-7.30 (m, 2H), 7.25-7.10 (m, 1H),6.85-6.70 (m, 3H), 5.00-4.70 (m, 1H), 4.32 (d, J=9.69 Hz, 1H), 4.10-3.95(m, 1H), 3.85-3.70 (m, 1H), 2.00-1.80 (m, 2H), 1.75-1.20 (m, 7H), 1.13(s, 3H), 0.95-0.75 (m, 1H), 0.81 (s, 9H); ESI-MS calculated for C₃₀H₃₇³⁵Cl₂FN₃O₃ [M+H]⁺: 576.2196. Found: 576.58.

MI-7102—TFA Salt

¹H NMR (300 MHz, MeOH-d4): 7.36-7.25 (m, 1H), 7.24-7.11 (m, 2H), 6.86(d, J=1.8 Hz, 1H), 6.80 (dd, J=1.8, 8.1 Hz, 1H), 6.72 (d, J=8.1 Hz, 1H),4.82 (d, J=9.6 Hz, 1H), 4.36 (d, J=9.6 Hz, 1H), 4.04 (dd, J=2.4, 7.4 Hz,1H), 3.74-3.56 (m, 1H), 3.56-3.40 (m, 1H), 2.05-1.78 (m, 5H), 1.75-1.59(m, 1H), 1.43-1.04 (m, 5H), 0.81 (s, 9H); ESI-MS calculated forC₂₉H₃₅ClF₂N₃O₃ (M+H)⁺ requires 546.23. found 546.58.

MI-7103—TFA Salt

¹H NMR (300 MHz, MeOH-d4): 8.38 (d, J=7.7 Hz, 1H), 7.54 (t, J=6.7 Hz,1H), 7.40 (d, J=7.1 Hz, 1H), 7.20 (t, J=7.9 Hz, 1H), 6.93 (d, J=6.1 Hz,1H), 6.86 (d, J=8.7 Hz, 1H), 4.45 (d, J=10.3 Hz, 1H), 4.13 (dd, J=2.8,7.5 Hz, 1H), 3.77-3.55 (m, 1H), 3.55-3.42 (m, 1H), 2.09-1.71 (m, 4H),1.70-1.56 (m, 1H), 1.45-1.02 (m, 5H), 0.82 (s, 9H); ESI-MS calculatedfor C₂₉H₃₄Cl₂F₂N₃O₃ (M+H)⁺ requires 580.19. found 580.67.

MI-7104—TFA Salt

¹H NMR (300 MHz, MeOH-d4): 7.49 (t, J=7.2 Hz, 1H), 7.45-7.38 (m, 1H),7.22 (t, J=8.0 Hz, 1H), 6.85-6.68 (m, 2H), 4.80 (d, J=9.8 Hz, 1H), 4.36(d, J=9.9 Hz, 1H), 4.01 (dd, J=2.4, 7.6 Hz, 1H), 3.74-3.57 (m, 1H),3.55-3.39 (m, 1H), 2.04-1.77 (m, 4H), 1.74-1.59 (m, 1H), 1.44-1.04 (m,5H), 0.90 (d, J=4.5 Hz, 1H), 0.82 (s, 9H); ESI-MS calculated forC₂₉H₃₄ClF₃N₃O₃ (M+H)⁺ requires 564.22. found 564.58.

MI-7105—TFA Salt

¹H NMR (300 MHz, MeOH-d4): 7.49 (t, J=7.2 Hz, 1H), 7.45-7.38 (m, 1H),7.22 (t, J=8.0 Hz, 1H), 6.85-6.68 (m, 2H), 4.80 (d, J=9.8 Hz, 1H), 4.36(d, J=9.9 Hz, 1H), 4.01 (dd, J=2.4, 7.6 Hz, 1H), 3.74-3.57 (m, 1H),3.55-3.39 (m, 1H), 2.04-1.77 (m, 4H), 1.74-1.59 (m, 1H), 1.44-1.04 (m,5H), 0.90 (d, J=4.5 Hz, 1H), 0.82 (s, 9H); ESI-MS calculated forC₂₉H₃₅ClF₂N₃O₃ (M+H)⁺ requires 546.23. found 546.58.

MI-7106—TFA Salt

¹H NMR (300 MHz, MeOH-d4): 8.36 (d, J=7.0 Hz, 1H), 7.59 (d, J=8.1 Hz,1H), 7.41-7.11 (m, 4H), 7.04 (d, J=7.6 Hz, 1H), 6.78 (d, J=1.8 Hz, 1H),5.19 (d, J=11.3 Hz, 1H), 4.44 (J=8.1 Hz, 1H), 4.07 (d, J=11.3 Hz, 1H),3.74-3.53 (m, 1H), 3.53-3.37 (m, 2.08-1.83 (m, 3H), 1.83-1.69 (m, 1H),1.61-1.44 (m, 1H), 1.44-1.08 (m, 4H), 1.07-0.72 (m, 1H), 0.88 (s, 9H);ESI-MS calculated for C₂₉H₃₆Cl₂N₃O₃ (M+H)⁺ requires 544.21. found544.67.

MI-7108—TFA Salt

¹H NMR (300 MHz, MeOH-d4/DMSO-d6): 10.15 (s, 1H), 7.76 (d, J=8.2 Hz,1H), 7.22 (s, 1H), 7.17-7.00 (m, 3H), 6.94 (d, J=7.1 Hz, 1H), 6.81 (d,J=6.0 Hz, 1H), 4.42 (d, J=8.3 Hz, 1H), 4.09 (d, J=3.0 Hz, 1H), 3.79 (d,J=8.3 Hz, 1H), 3.73-3.49 (m, 2H), 3.35 (d, J=9.5 Hz, 1H), 2.10-1.84 (m,4H), 1.52-1.11 (m, 5H), 0.87 (s, 9H); ¹³C NMR (75 MHz, MeOH-d4/DMSO-d6):177.1, 172.4, 153.6 (d, J_(C-F)=242.7 Hz), 138.7, 138.5 (d, J_(C-F)=2.4Hz), 133.2, 129.0, 127.544, 127.541 (d, J_(C-F)=6.7 Hz), 126.8, 126.5,119.7 (d, J_(C-F)=19.2 Hz), 111.3, 110.4 (d, J_(C-F)=24.1 Hz), 68.4,66.6, 65.7, 64.0, 58.6, 46.8, 42.2, 33.26, 33.20, 30.4, 30.2, 29.7,29.5; ESI-MS calculated for C₂₉H₃₅Cl₂FN₃O₃ (M+H)+ requires 562.20. found562.67.

MI-7109—TFA Salt

¹H NMR (300 MHz, MeOH-d4): 7.47 (t, J=6.7 Hz, 1H), 7.42-7.33 (m, 1H),7.18 (t, J=7.7 Hz, 1H), 6.87 (d, J=1.8 Hz, 1H), 6.78 (dd, J=1.8, 8.1 Hz,1H), 6.70 (d, J=8.1 Hz, 1H), 4.40 (d, J=9.7 Hz, 1H), 4.11 (dd, J=2.5,7.6 Hz, 1H), 2.77-2.65 (m, 1H), 1.99 (dd, J=7.6, 15.3 Hz, 1H), 1.24 (dd,J=2.5, 15.3 Hz, 1H), 0.92-0.62 (m, 2H), 0.81 (s, 9H), 0.56-0.30 (m, 2H);ESI-MS calculated for C₂₆H₂₉Cl₂FN₃O₂ (M+H)⁺ requires 504.16. found504.58.

B049—TFA Salt

¹H NMR (300 MHz, CD₃OD): 7.597 (d, J=8.73 Hz, 1H), 7.30-7.10 (m, 3H),7.10-7.00 (m, 1H), 6.84 (d, J=6.14 Hz, 1H), 5.35 (d, J=12.85 Hz, 1H),4.40 (dd, J=7.62, 3.80 Hz, 1H), 4.15 (d, J=12.92 Hz, 1H), 1.71 (dd,J=15.32, 7.68 Hz, 1H), 1.12 (dd, J=15.32, 3.79 Hz, 1H), 0.92 (s, 9H);ESI-MS calculated for C₂₃H₂₄ ³⁵Cl₂FN₂O₃ [M+H]⁺: 465.1148. Found: 465.50.

B053—TFA Salt

¹H NMR (300 MHz, CD₃OD): 7.67 (d, J=8.41 Hz, 1H), 7.35-7.18 (m, 3H),7.10-7.00 (m, 1H), 6.87 (d, J=6.03 Hz, 1H), 5.62 (d, J=12.10 Hz, 1H),4.42 (dd, J=8.54, 1.92 Hz, 1H), 4.30-4.15 (m, 2H), 4.23 (d, J=12.00 Hz,1H), 1.96 (dd, J=15.44, 8.61 Hz, 1H), 1.24-1.15 (m, 1H), 1.13 (t, J=7.13Hz, 3H), 0.91 (s, 9H); ESI-MS calculated for C₂₅H₂₈ ³⁵Cl₂FN₂O₃ [M+H]⁺:493.1461. Found: 493.30.

B059—TFA Salt

¹H NMR (300 MHz, CD₃OD): 7.45-7.34 (m, 1H), 7.26-7.12 (m, 1H), 7.04-6.93(m, 1H), 6.90 (d, J=1.80 Hz, 1H), 6.65 (dd, J=8.08, 1.80 Hz, 1H), 4.41(d, J=9.25 Hz, 1H), 3.96 (quint, J=8.13 Hz, 1H), 2.51-2.07 (m, 2H),2.40-2.20 (m, 2H), 1.88 (dd, J=14.20, 9.91 Hz, 1H), 1.32 (s, 3H),1.20-0.80 (m, 1H), 0.88 (s, 9H); ¹³C NMR (75 MHz, CD₃OD): 181.3, 172.9(d, J_(C-F)=266.9 Hz), 168.6, 162.7, 145.3, 135.8, 131.7, 130.7 (d,J_(C-F)=38.6 Hz), 126.2 (d, J_(C-F)=4.5 Hz), 126.1, 123.6, 122.9, 122.7,111.4, 78.4, 67.7, 63.4, 46.0, 45.8, 44.3, 38.0, 31.4, 30.2, 27.6;ESI-MS calculated for C₂₈H₃₁ ³⁵Cl₂FN₃O₃ [M+H]⁺: 546.1727. Found: 546.50.

B066-15—TFA Salt

¹H NMR (300 MHz, CD₃OD): 7.60-7.45 (m, 1H), 7.45-7.35 (m, 1H), 7.22 (t,J=7.96 Hz, 1H), 6.90 (d, J=1.80 Hz, 1H), 6.75 (dd, J=8.14, 1.92 Hz, 1H),6.41 (d, J=8.13 Hz, 1H), 5.07 (d, J=8.40 Hz, 1H), 4.39 (d, J=8.40 Hz,1H), 4.01 (dd, J=7.76, 2.17 Hz, 1H), 1.95 (dd, J=15.35, 7.83 Hz, 1H),1.12 (dd, J=15.35, 2.17 Hz, 1H), 0.82 (s, 9H); ESI-MS calculated forC₂₃H₂₄ ³⁵Cl₂FN₂O₃ [M+H]⁺: 465.1148. Found: 465.42.

MI-519-72—TFA Salt

ESI-MS calculated for C₂₈H₃₂ ³⁵Cl₂F₃N₄O₂ [M+H]⁺: 583.1854. Found:583.42.

MI-519-75—TFA Salt

¹H NMR (300 MHz, CD₃OD): 7.64-7.54 (m, 1H), 7.42-7.60 (m, 1H), 7.26-7.18(m, 1H), 6.88 (s, 1H), 6.80-6.70 (m, 1H), 6.50-6.40 (m, 1H), 5.20-5.00(m, 1H), 4.80-4.30 (m, 4H), 4.30-4.10 (m, 2H), 4.10-4.00 (m, 1H), 3.98(s, 3H), 1.90 (dd, J=14.96, 7.33 Hz, 1H), 1.11 (d, J=14.96 Hz, 1H), 0.81(s, 9H); ESI-MS calculated for C₂₇H₃₂ ³⁵Cl₂FN₄O₂ [M+H]⁺: 533.1886.Found: 533.58.

MI-519-76—TFA Salt

ESI-MS calculated for C₂₈H₃₃ ³⁵Cl₂F₂N₄O₂ [M+H]⁺: 565.1949. Found:565.42.

MI-519-77—TFA Salt

¹H NMR (300 MHz, CD₃OD): 7.50-7.40 (m, 1H), 7.40 (m, 1H), 7.20-7.10 (m,1H), 6.85 (d, J=1.40 Hz, 1H), 6.84-6.72 (m, 2H), 5.00-4.80 (m, 1H), 4.45(d, J=10.10 Hz, 1H), 4.02 (t, J=6.61 Hz, 1H), 3.90 (quintet, J=8.07 Hz,1H), 2.50-2.25 (m, 2H), 2.10-1.82 (m, 3H), 1.81-1.31 (m, 8H), 1.30 (s,3H), 1.10-0.91 (m, 1H), 0.91-0.81 (m 1H); ESI-MS calculated for C₂₉H₃₃³⁵Cl₂FN₃O₃ [M+H]⁺: 560.1883. Found: 560.50.

MI-519-78—TFA Salt

¹H NMR (300 MHz, CD₃OD): 7.45-7.31 (m, 2H), 7.20-7.11 (m, 1H), 6.86-6.82(m, 1H), 6.81-6.78 (m, 2H), 4.90-4.80 (m, 1H), 4.45 (d, J=10.33 Hz, 1H),4.10-3.95 (m, 1H), 3.70-3.60 (m, 1H), 3.50-3.40 (m, 1H), 2.10-1.05 (m,17H), 1.05-0.95 (m, 1H), 0.95-0.80 (m, 1H); ESI-MS calculated for C₃₀H₃₅³⁵Cl₂FN₃O₃ [M+H]⁺: 574.2040. Found: 574.58.

MI-519-79—TFA Salt

¹H NMR (300 MHz, CD₃OD): 7.78 (d, J=6.92 Hz, 1H), 7.56-7.42 (m, 2H),6.90 (d, J=1.89 Hz, 1H), 6.68 (dd, J=8.15, 1.84 Hz, 1H), 6.03 (dd,J=8.13, 3.66 Hz, 1H), 4.80-4.70 (m, 1H), 4.58 (d, J=5.51 Hz, 1H),4.10-3.70 (m, 7H), 3.54 (dt, J=15.10, 5.20 Hz, 2H), 3.40-3.20 (m, 3H),1.79 (ddd, J=14.60, 8.49, 1.59 Hz, 1H), 1.00-0.80 (m, 1H), 0.81 (s, 9H);ESI-MS calculated for C₂₉H₃₆ ³⁵Cl₃N₄O₃ [M+H]⁺: 593.1853. Found: 593.75.

MI-519-80—TFA Salt

¹H NMR (300 MHz, CD₃OD): 7.80-7.72 (m, 1H), 7.50-7.38 (m, 2H), 6.87 (d,J=1.81 Hz, 1H), 6.71 (dd, J=8.16, 1.81 Hz, 1H), 6.52-6.40 (m, 1H),4.96-4.80 (m, 1H), 4.62 (d, J=8.69 Hz, 1H), 4.10-3.95 (m, 1H), 3.70-3.55(m, 1H), 3.50-3.45 (m, 1H), 2.00-1.80 (m, 3H), 1.80-1.60 (m, 1H),1.40-1.00 (m, 5H), 0.95-0.85 (m, 1H), 0.80 (s, 9H); ESI-MS calculatedfor C₂₉H₃₅ ³⁵Cl₃N₃O₃ [M+H]⁺: 578.1744. Found: 578.75.

C02701—TFA Salt

¹H NMR (300 MHz, MeOH-d4): 8.82 (d, J=6.83 Hz, 1H), 7.65-7.55 (m, 1H),7.45-7.30 (m, 1H), 7.20-7.05 (m, 3H), 6.80-6.75 (m, 1H), 5.40-5.10 (m,1H), 4.61 (d, J=11.39 Hz, 1H), 4.50 (d, J=7.66 Hz, 1H), 3.95-3.80 (m,1H), 2.45-2.30 (m, 1H), 2.30-2.15 (m, 1H), 2.05-1.80 (m, 2H), 1.80-1.60(m, 1H), 1.27 (s, 3H), 1.20-1.08 (m, 1H), 0.86 (s, 9H); ¹³C NMR (75 MHz,MeOH-d4): 177.8, 167.0, 160.0-148.0 (m, 2×C_(sp2)-F), 145.2, 137.2,126.8, 126.5-126.0 (m), 125.0, 124.1, 123.5, 122.1 (d, J_(C-F)=9.74 Hz),119.1 (d, J_(C-F)=17.1 Hz), 112.1, 67.3, 64.5, 64.2, 62.6, 48.5, 45.6,45.5, 43.3, 38.3, 31.0, 29.5, 27.5; ESI-MS calculated for C₂₈H₃₃³⁵ClF₂N₃O₃[M+H]⁺: 532.2179. Found: 532.50.

C02901—TFA Salt

¹H NMR (300 MHz, MeOH-d4): 7.80-7.65 (m, 1H), 7.60-7.50 (m, 1H),7.40-7.30 (m, 1H), 7.20-7.10 (m, 1H), 6.80-6.65 (m, 1H), 5.50-5.10 (m,1H), 4.60 (d, J=11.39 Hz, 1H), 4.50 (d, J=6.96 Hz, 1H), 3.95-3.80 (m,1H), 2.50-2.30 (m, 1H), 2.30-2.20 (m, 1H), 2.10-1.80 (m, 2H), 1.80-1.65(m, 1H), 1.27 (s, 3H), 1.20-1.05 (m, 1H), 0.87 (s, 9H); ¹³C NMR (75 MHz,MeOH-d4): 177.8, 167.0, 160.0-145.0 (m, 3×C_(sp2)-F), 132.6, 128.6,126.6, 122.5 (d, J_(C-F)=18.9 Hz), 121.3 (d, J_(C-F)=13.0 Hz), 118.8,115.4 (d, J_(C-F)=21.7 Hz), 115.1, 101.8 (d, J_(C-F)=23.3 Hz), 67.3,64.6, 64.3, 62.5, 48.7, 45.6, 45.5, 43.4, 38.3, 31.0, 29.5, 27.5; ESI-MScalculated for C₂₈H₃₂ ³⁵ClF₃N₃O₃ [M+H]⁺: 550.2084. Found: 550.33.

C03001—TFA Salt

¹H NMR (300 MHz, MeOH-d4): 7.70 (d, J=7.30 Hz, 1H), 7.60-7.50 (m, 1H),7.45-7.35 (m, 1H), 7.25-7.15 (m, 1H), 6.88 (d, J=6.00 Hz, 1H), 5.21 (d,J=11.35 Hz, 1H), 4.61 (d, J=11.37 Hz, 1H), 4.53 (d, J=8.19 Hz, 1H),3.95-3.80 (m, 1H), 2.50-2.35 (m, 1H), 2.35-2.15 (m, 1H), 2.00-1.80 (m,2H), 1.80-1.60 (m, 1H), 1.29 (s, 3H), 1.25-1.05 (m, 1H), 0.89 (s, 9H);¹³C NMR (75 MHz, MeOH-d4): 177.3, 166.7, 157.6 (d, J_(C-F)=249.5 Hz),155.7 (d, J_(C-F)=243.5 Hz), 140.4 (d, J_(C-F)=2.8 Hz), 132.5, 128.4,126.4 (d, J_(C-F)=4.9 Hz), 125.0 (d, J_(C-F)=7.4 Hz), 123.4 (d,J_(C-F)=19.5 Hz), 122.3 (d, J_(C-F)=18.9 Hz), 121.0 (d, J_(C-F)=13.0Hz), 114.5 (d, J_(C-F)=25.1 Hz), 104.8, 67.1, 64.6, 64.2, 62.4, 47.3,45.4, 45.3, 43.2, 38.2, 30.8, 29.2, 27.3; ESI-MS calculated for C₂₈H₃₂³⁵Cl₂F₂N₃O₃ [M+H]⁺: 566.1789. Found: 566.50.

C03401—TFA Salt

¹H NMR (300 MHz, MeOH-d4): 7.58 (d, J=8.07 Hz, 1H), 7.30-7.10 (m, 4H),7.02 (d, J=7.67 Hz, 1H), 6.77 (d, J=1.54 Hz, 1H), 5.40-5.20 (m, 1H),4.44 (d, J=7.09 Hz, 1H), 4.10 (d, J=11.25 Hz, 1H), 3.95-3.80 (m, 1H),2.45-2.30 (m, 1H), 2.30-2.15 (m, 1H), 2.05-1.85 (m, 2H), 1.80-1.70 (m,1H), 1.27 (s, 3H), 1.20-1.10 (m, 1H), 0.86 (s, 9H); ¹³C NMR (75 MHz,MeOH-d4): 177.8, 167.3, 145.3, 137.1, 135.8, 134.4, 131.4, 130.4, 129.5,128.3, 126.3, 124.2, 124.1, 112.2, 67.3, 64.9, 64.2, 62.8, 57.2, 45.7,45.6, 43.4, 38.3, 31.0, 29.5, 27.5; ESI-MS calculated for C₂₈H₃₄³⁵Cl₂N₃O₃[M+H]⁺: 530.1977. Found: 530.58.

C03701—TFA Salt

¹H NMR (300 MHz, MeOH-d4): 9.00-8.80 (m, 1H), 7.73 (d, J=8.42 Hz, 1H),7.40-7.20 (m, 3H), 7.15-7.05 (m, 1H), 6.89 (d, J=6.00 Hz, 1H), 5.32 (d,J=11.34 Hz, 1H), 4.52 (d, J=7.91 Hz, 1H), 4.20 (d, J=11.28 Hz, 1H),4.00-3.80 (m, 1H), 2.50-2.35 (m, 1H), 2.35-2.20 (m, 1H), 2.20-1.90 (m,2H), 1.90-1.80 (m, 1H), 1.31 (s, 3H), 1.30-1.15 (m, 1H), 0.91 (s, 9H);¹³C NMR (75 MHz, MeOH-d4): 178.8, 168.2, 157.3 (d, J_(C-F)=255.8 Hz),142.1 (d, J_(C-F)=2.6 Hz), 137.1, 135.5, 132.8, 131.7, 130.7, 129.6,127.2 (d, J_(C-F)=7.2 Hz), 124.7 (d, J_(C-F)=19.3 Hz), 115.5 (d,J_(C-F)=24.9 Hz), 114.8, 68.6, 66.6, 65.3, 64.0, 58.2, 47.0, 46.8, 44.7,39.5, 32.2, 30.8, 28.8; ESI-MS calculated for C₂₈H₃₃ ³⁵Cl₂FN₃O₃ [M+H]⁺:548.1883. Found: 548.42.

C04801—TFA Salt

¹H NMR (300 MHz, MeOH-d4): 9.00-8.80 (m, 1H), 7.70 (d, J=8.35 Hz, 1H),7.50-7.35 (m, 1H), 7.30-7.10 (m, 2H), 6.88 (d, J=6.88 Hz, 1H), 5.30 (d,J=11.32 Hz, 1H), 4.66 (d, J=11.33 Hz, 1H), 4.56 (d, J=7.43 Hz, 1H),4.00-3.80 (m, 1H), 2.50-2.35 (m, 1H), 2.35-2.20 (m, 1H), 2.10-1.90 (m,2H), 1.80-1.70 (m, 1H), 1.30 (s, 3H), 1.16 (d, J=15.34 Hz, 1H), 0.90 (s,9H); ¹³C NMR (75 MHz, MeOH-d4): 177.5, 166.9, 160-145 (m, 2×C_(sp2)-F),155.9 (d, J_(C-F)=243.4 Hz), 140.7 (d, J_(C-F)=2.69 Hz, 1H), 126.5-126.1(m), 125.6 (d, J_(C-F)=7.6 Hz), 125.0 (d, J_(C-F)=3.4 Hz), 123.6 (d,J_(C-F)=19.5 Hz), 122.0 (d, J_(C-F)=9.8 Hz), 119.1 (d, J_(C-F)=17.1 Hz),114.7 (d, J_(C-F)=25.0 Hz), 113.4, 67.3, 64.7, 64.3, 62.5, 48.2, 45.6,45.6, 43.4, 38.3, 31.0, 29.5, 27.5; ESI-MS calculated for C₂₈H₃₂³⁵ClF₃N₃O₃ [M+H]⁺: 550.2084. Found: 550.42.

MI-710201—TFA Salt

¹H NMR (300 MHz, MeOH-d4): 7.57 (d, J=8.0 Hz, 1H), 7.50-7.36 (m, 1H),7.27-7.07 (m, 3H), 6.79 (s, 1H), 5.11 (d, J=11.1 Hz, 1H), 4.55 (d,J=11.0 Hz, 1H), 4.39 (d, J=7.7 Hz, 1H), 3.71-3.52 (m, 1H), 3.52-3.37 (m,1H), 3.21 (dd, J=7.4, 14.5 Hz, 1H), 1.92 (d, J=9.6 Hz, 1H), 1.86-1.70(m, 2H), 1.58 (d, J=11.8 Hz, 1H), 1.43-1.18 (m, 4H), 1.12 (d, J=15.5 Hz,1H), 0.99 (d, J=13.0 Hz, 1H), 0.88 (s, 9H); ESI-MS calculated forC₂₉H₃₅ClF₂N₃O₃ (M+H)⁺ requires 546.23. found 546.58.

MI-710401—TFA Salt

¹H NMR (300 MHz, MeOH-d4): 7.67 (t, J=8.6 Hz, 1H), 7.57 (t, J=6.8 Hz,1H), 7.38 (t, J=6.8 Hz, 1H), 7.15 (t, J=7.8 Hz, 1H), 6.71 (dd, J=6.6,10.1 Hz, 1H), 5.01 (d, J=10.8 Hz, 1H), 4.52 (d, J=10.8 Hz, 1H),4.40-4.21 (m, 1H), 3.74-3.56 (m, 1H), 3.56-3.40 (m, 1H), 2.08-1.87 (m,2H), 1.87-1.68 (m, 2H), 1.68-1.53 (m, 1H), 1.45-1.18 (m, 3H), 1.17-0.97(m, 2H), 0.89 (s, 9H); ESI-MS calculated for C₂₉H₃₄ClF₃N₃O₃ (M+H)⁺requires 564.22. found 564.58.

MI-710501—TFA Salt

¹H NMR (300 MHz, MeOH-d4): 7.66-7.53 (m, 1H), 7.44-7.33 (m, 1H),7.22-7.09 (m, 1H), 6.93-6.79 (m, 1H), 6.59-6.51 (m, 1H), 5.40-5.31 (m,1H), 4.63-4.48 (m, 1H), 4.41-4.30 (m, 1H), 2.41-2.20 (m, 2H), 2.15-1.97(m, 2H), 1.95-1.85 (m, 1H), 1.85-1.71 (m, 1H), 1.71-1.47 (m, 3H),1.19-1.07 (m, 1H), 0.88 (s, 9H); ESI-MS calculated for C₂₉H₃₄ClF₃N₃O₃(M+H)⁺ requires 546.23. found 546.58.

MI-710901—TFA Salt

¹H NMR (300 MHz, MeOH-d4): 7.61 (d, J=8.1 Hz, 1H), 7.53 (t, J=6.7 Hz,1H), 7.40 (t, J=7.0 Hz, 1H), 7.21-7.08 (m, 2H), 6.79 (d, J=1.6 Hz, 1H),5.14 (d, J=11.3 Hz, 1H), 4.60 (d, J=11.3 Hz, 1H), 4.48 (d, J=7.0 Hz,1H), 2.78-2.58 (m, 1H), 1.86 (dd, J=8.4, 15.4 Hz, 1H), 1.13 (d, J=15.4Hz, 1H), 0.88 (s, 9H), 0.78-0.60 (m, 2H), 0.47-0.16 (m, 2H); ¹³C NMR (75MHz, MeOH-d4): 177.9, 169.5, 157.8 (d, J_(C-F)=249.4 Hz), 145.2, 137.2,132.6, 128.7, 126.8 (d, J_(C-F)=1.6 Hz), 126.6 (d, J_(C-F)=4.9 Hz),124.2, 123.6, 122.5 (d, J_(C-F)=18.8 Hz), 121.8 (d, J_(C-F)=13.1 Hz),64.7, 64.4, 62.9, 43.5, 31.0, 29.6, 23.9, 6.7, 6.5; ESI-MS calculatedfor C₂₆H₂₉Cl₂FN₃O₂ (M+H)⁺ requires 504.16. found 504.58.

Example 2 Fluorescence-Polarization MDM2 Binding Assay

The binding affinity of the MDM2 inhibitors was determined using anoptimized, sensitive and quantitative fluorescence polarization-based(FP-based) binding assay using a recombinant human His-tagged MDM2protein (residues 1-118) and a fluorescently tagged p53-based peptide.

The design of the fluorescence probe was based upon a previouslyreported high-affinity p53-based peptidomimetic compound(5-FAM-βAla-βAla-Phe-Met-Aib-pTyr-(6-Cl-LTrp)-Glu-Ac3c-Leu-Asn-NH2 (SEQID NO: 1)) García-Echeverría et al., J. Med. Chem. 43: 3205-3208(2000)). This tagged peptide is called PMDM6-F. The K_(d) value ofPMDM6-F with the recombinant MDM2 protein was determined from thesaturation curve. MDM2 protein was serially double diluted in a Dynex96-well, black, round-bottom plate, and the PMDM6-F peptide was added at1 nM concentration. The assay was performed in the buffer: 100 mMpotassium phosphate, pH 7.5; 100 μg/mL bovine gamma globulin; 0.02%sodium azide, 0.01% Triton X-100) and the polarization values weremeasured after 3 h of incubation using an ULTRA READER (Tecan U.S. Inc.,Research Triangle Park, N.C.). The IC₅₀ value was obtained by fittingthe mP values in a sigmoidal dose-response curve (variable slope) with anon-linear regression, and was determined to be 1.40 nM±0.25. The K_(d)value was calculated using the equation: K_(d) value=IC₅₀−L0/2. L0/2 isthe concentration of the free ligand (PMDM6-F). Since PMDM6-F was usedat a final concentration of 1 nM, L0/2 was 0.5 nM.

Dose-dependent, competitive binding experiments were performed withserial dilutions of a tested compound in DMSO. A 5 μL sample of thetested compound and pre-incubated MDM2 protein (10 nM) and PMDM6-Fpeptide (1 nM) in the assay buffer (100 mM potassium phosphate, pH 7.5;100 μg/mL bovine gamma globulin; 0.02% sodium azide, 0.01% TritonX-100), were added in a Dynex 96-well, black, round-bottom plate toproduce a final volume of 125 pt. For each assay, the controls includedthe MDM2 protein and PMDM6-F (equivalent to 0% inhibition), PMDM6-Fpeptide alone (equivalent to 100% inhibition). The polarization valueswere measured after 3 h of incubation. The IC₅₀ values, i.e. theinhibitor concentration at which 50% of bound peptide is displaced, weredetermined from a plot using nonlinear least-squares analysis. Curvefitting was performed using GRAPHPAD PRISM software (GraphPad Software,Inc., San Diego, Calif.). As presented in Tables 19A-D and FIGS. 23 and24, compounds provided herein are potent MDM2 antagonists. Compoundsshown in Tables 19A-D as the free base were tested either as the freebase or as the CF₃CO₂H (TFA) or HCl salt. In general, comparable assayresponses are expected between the free base and salt form of acompound.

Example 3 Cell Growth Assay

Isogenic HCT-116 colon cancer cell lines were a kind gift from Prof.Bert Vogelstein (Johns Hopkins, Baltimore, Md.) and were maintained inMcCoy's 5A medium containing 10% FBS. All other cell lines were obtainedfrom ATCC, (Manassas, Va.) and were maintained in RPMI-1640 mediumcontaining 10% FBS.

Cells were seeded in 96-well flat bottom cell culture plates at adensity of 2−3×10³ cells/well with compounds and incubated for 4 days.The rate of cell growth inhibition after treatment with increasingconcentrations of the tested compounds was determined by WST-8(2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazoliummonosodium salt (Dojindo Molecular Technologies Inc., Gaithersburg,Md.). WST-8 was added at a final concentration of 10% to each well, andthen the plates were incubated at 37° C. for 2-3 hrs. The absorbance ofthe samples was measured at 450 nm using a TECAN ULTRA Reader. Theconcentration of the compounds that inhibited cell growth by 50% (IC₅₀)was calculated by comparing absorbance in the untreated cells and thecells treated with the compounds using the GraphPad Prism software(GraphPad Software, La Jolla, Calif. 92037, USA). The results of thisassay are presented in Tables 19A-D.

TABLE 19A IC₅₀ (μM) SJSA-1 HCT-116 LNCAP PC-3 (p53 wild- (p53 wild- (p53wild- HCT-116 (deleted Example Chemical Structure MDM2 type) type) type)(p53-/-) p53) MI-219-M1 (a)

<1 NT <3 <3 >10 >10 MI-219-M1 (b)

<1 NT <3 <3 >10 >10 MI-519-24

<2 NT <2 <3 6.5 ± 0.7 3.7 ± 0.2 MI-519-27

<1 <3 <2 <2 >10 >5 MI-519-28

<2 NT <2 <2 >5  4.1 ± 0.2 MI-519-29

<1 NT <5 <5 >10 >10 MI-519-30

<2 NT 1.3 ± 0.3 1.1 ± 0.0 19.6 ± 5.2  19.1 ± 2.4  MI-519-31 (isomer ofMI-519-30)

<2 NT 2.0 ± 0.8 1.3 ± 0.2 20.1 ± 2.5  24.7 ± 2.0  MI-519-35

<1 NT 1.8 ± 0.5 1.4 ± 0.5 5.3 ± 0.6 6.7 ± 0.9 MI-519-36

<1 NT 1.2 ± 0.6 0.9 ± 0.1 29.8 ± 6.5  20.6 ± 5.3  MI-519-37

<1 NT 1.0 ± 0.5 0.6 ± 0.1 14.9 ± 2.0  14.0 ± 2.2  MI-519-38

<1 2.1 ± 0.1 1.4 ± 0.6 1.0 ± 0.3 5.3 ± 1.6 3.1 ± 0.4 MI-519-40

<1 1.3 ± 0.3 1.0 ± 0.1 1.0 ± 0.1 18.5 ± 5.5  14.6 ± 2.7  MI-519-41

<1 1.3 ± 0.4 1.0 ± 0.3 1.2 ± 0.0 17.9 ± 2.2  14.9 ± 1.1  MI-519-43

 <10 0.9 ± 0.1 0.9 ± 0.0 1.0 ± 0.0 4.1 ± 0.5 3.4 ± 0.0 MI-519-44

 <10 0.9 ± 0.1 0.8 ± 0.0 1.3 ± 0.2 2.8 ± 0.7 2.6 ± 0.1 MI-519-45

 <10 0.8 ± 0.2 0.9 ± 0.3 1.0 ± 0.2 6.9 ± 1.9 5.1 ± 0.9 MI-519-46

<1 0.5 ± 0.3 0.4 ± 0.1 0.5 ± 0.3 4.3 ± 0.4 3.7 ± 0.4 MI-519-47

 <10 1.1 ± 0.0 1.1 ± 0.2 1.1 ± 0.3 7.8 ± 0.9 5.7 ± 0.6 MI-519-49

<1 NT 0.84 0.85 12.7  8.1 MI-519-50

<1 1.7 ± 1.7 0.9 ± 0.2 1.3 ± 0.4 23.71 16.9 ± 4.5  MI-519-51

<1 0.6 ± 0.2 0.6 ± 0.3 0.6 ± 0.2 13.4 ± 0.8  8.2 ± 2.5 MI-519-51- epi

<1 NT NT <2 NT >10 MI-519-56

<1 <1 <1  >10 MI-519-60

<1 <3 <3 <3 NT NT MI-519-61

<1 <3 <3 <3 NT NT MI-519-62

<1 <3 <3 <3 >10 >10 MI-519-63

<1 2.1 2.0 2.3 NT NT MI-519-64

<1 <1 <1 <1 >10 NT MI-519-65

<1 <3 <3 <3 >10 NT MI-519-68

NT NT NT NT NT NT MI-519-69

NT <5 <5 <5 >10 >10 MI-519-70

NT <5 <5 <5 >10 >10 MI-748

<1 NT 0.9 ± 0.7 0.8 ± 0.0 7.6 ± 1.0 4.1 ± 0.1 MI-749

<1 NT 1.2 ± 0.4 1.0 ± 0.1 7.8 ± 1.3 5.0 ± 0.5 MI-751

<1 NT 1.0 ± 0.2 0.7 ± 0.2 6.2 ± 1.2 3.9 ± 0.9 MI-758

<1 NT 0.6 ± 0.0 0.6 ± 0.0 19.1 ± 3.6  18.7 ± 7.1  MI-764

<1 1.2 ± 0.3 0.5 ± 0.2 1.0 ± 0.4 3.8 ± 0.7 2.4 ± 0.4 MI-765

 NT. 1.4 ± 0.1 1.1 ± 0.2 1.5 ± 0.9 4.7 ± 2.0 2.6 ± 0.2 MI-771

<1 1.2 ± 0.3 1.0 ± 0.3 1.1 ± 0.1 12.0 ± 1.1  13.7 ± 4.4  MI-772

<1 <1 <1 <1 >5 >5 MI-773

<1 <1 <1 <1 >10 >10 MI-779

  <0.1 1.0 2.5 1.3 13.6 11.6  MI-781

  <0.1 0.7 0.4 NT 10.1 NT MI-739

<5 NT 2.1 ± 0.6 1.5 ± 0.5 3.0 ± 0.2 3.2 ± 0.7 MI-740

<5 NT 2.8 ± 0.1 2.0 ± 0.3 5.4 ± 1.9 4.8 ± 0.8 MI-742

 <10 NT 1.7 ± 0.1 2.2 ± 0.3 19.9 ± 10.3 10.2 ± 4.8  MI-744

 <10 NT 2.4 ± 0.2 1.7 ± 0.1 5.7 ± 3.4 7.7 ± 3.1 MI-746

 <10 NT 2.3 ± 0.1 2.2 ± 0.4 6.7 ± 1.8 6.9 ± 1.2 MI-747

 NT. NT 1.3 1.3 3.9 3.7 MI-759

<1 NT 1.4 ± 0.6 1.4 ± 0.1 5.2 ± 1.0 5.5 ± 2.3 MI-766

<1 1.7 ± 1.4 0.7 ± 0.0 0.9 ± 0.0 6.9 ± 1.4 5.9 ± 2.2 MI-767

<1 0.9 ± 0.6 0.7 ± 0.2 0.9 ± 0.2 2.4 ± 1.0 2.2 ± 0.1 MI-768

<1 0.8 ± 0.1 0.5 ± 0.2 0.7 ± 0.2 9.8 ± 0.4 7.3 ± 0.8 MI-769

<1 0.6 1.6 1.5 10.2 13.0  MI-774

  <0.1 0.3 ± 0.1 0.3 ± 0.1 0.3  5.6 NT MI-776

<1 3.2 2.7 2.4 NT NT MI-782

<1 1.2 0.7 NT  4.7 NT MI-783

  <0.5 1.1 0.9 NT 18.5 NT MI-784

  <0.5 0.7 0.4 NT 10.3 NT MI-785

  <0.5 0.7 0.5 NT  8.0 NT MI-786

<1 0.8 0.5 1.2 NT 14.4  MI-777

<5 9.2 8.9 6.6  7.8 MI-780

<3 <5 C027

<1 <3 <3 <3 >10 >10 C029

<5 <3 <3 <3 >5  >5 C031

<5 <3 <3 <3 >10 >10 C034

<1 <3 <3 <3 >10 >10 C035

<3 <5 <5 <5 >10 >10 C086

<3 MI-7102

<3 <3 <3 <3 >10 >10 MI-7104

<3 <3 <3 <3 >10 >10 MI-7105

<3 <3 <3 <3 >10 >10 MI-789

<3 <3 <3 <3 >10 >10 MI-790

<3 <3 <3 <3 >10 >10 MI-791

<3 <3 <3 <3 >10 >10 MI-792

<5 <10 <10 <10 >20 >20 MI-909

<1 <3 <3 <3 >5  >5  MI-911

<3 <5 <5 <5 >10 >10 MI-912

<3 <5 <5 <5 >10 >5  MI-913

<3 <5 <5 <5  8.8 4.9 MI-914

<1 <3 <3 <3 >10 >5  MI-915

<3 <10 <10 <10 >30 >30 MI-916

  <0.1 <5 <3 <3 >5  >5  MI-917

<1 1.4 <3 <3 <3 >10 >5  MI-901

<1 <3 <3 <3 >10 >5  MI-902

<3 <3 <3 <3  5.9 4.0 MI-903

<3 <3 <3 <3 12.1 3.5 MI-904

<1 <3 <3 <3  6.1 3.1 MI-910

<1 <3 <3 <3  9.8 7.0 MI-905

<1 <3 <3 <3  8.0 3.9 MI-906

<1 <3 <3 <3  9.1 3.6 MI-907

<1 <3 <3 <3 7  3.0 MI-908

 >10 22.5 5.3 4.3 >30 ~30 MI-519- 6401

  <0.1 0.4 0.1 0.3 12.2 10.5  MI-77301

  <0.1 0.8 0.1 0.1 12.9 13.0  C02701

  <0.1 0.2 0.2 0.3 13.2 12.9  C02901

  <0.1 0.5 0.6 0.2 16.4 15.6  C03001

  <0.1 0.2 0.2 0.1 14.2 29.8  C03401

  <0.1 0.3 0.6 0.3 17.3 17.4  C03701

  <0.1 0.7 0.2 0.4 21.0 26.7  C08301

  <0.1 C08601

  <0.5 C091

  <0.1 C096

  <0.1 MI-710201

  <0.1 0.6 0.3 0.3 27.6 19.3  MI-710301

  <0.1 0.1 0.1 0.1 11.9 12.5  MI-710401

  <0.5 0.8 0.4 0.4 13.0 12.7  MI-710601

  <0.1 0.4 0.3 0.2 11.5 18.4 

TABLE 19B Binding affinities to MDM2 protein as deter- mined by FPInhibition of cell growth in different cancer based cell lines (IC₅₀,μM) assay HCT116 HCT116 ID Structure MW IC₅₀ [μM] LNCaP PC3 p53 WT p53KO MI-701

622.94 >10  3.0 ± 3.5 48.9 ± 22.4 16.7 ± 13.0 33.8 ± 14.6 MI-702

560.87 >1  1.9 ± 0.3 14.3 ± 2.9 5.8 ± 1.7 12.7 ± 2.3  MI-703

628.97 >1  0.2 ± 0.0  3.9 ± 0.5 1.0 ± 0.2 4.3 ± 1.6 MI-704

636.97 >1  6.8 ± 0.2 18.0 ± 5.1 3.0 ± 0.9 12.1 ± 1.0  MI-705

588.93 <10  1.4 ± 0.8 43.9 ± 31.7 4.4 ± 4.0 32.0 ± 33.3 MI-706

572.88 <10  0.3 ± 0.0  4.6 ± 2.9 0.6 ± 0.1 1.3 ± 0.2 MI-707

581.49 <5  0.3 ± 0.0  4.6 ± 2.9 0.6 ± 0.1 1.3 ± 0.2 MI-708A

602.95 >3  3.5 ± 0.0 83.8 ± 11.3 3.8 ± 1.0 95.6 ± 18.2 MI-708B

602.95 >3  6.0 ± 0.3 84.5 ± 18.3 6.6 ± 2.2 96.9 ± 17.8 MI-709

651.00 >10  5.6 ± 1.1  7.2 ± 1.4 9.9 ± 1.7 9.4 ± 1.2 MI-710

643.00 (4.18) >1  1.2 ± 0.2 12.4 ± 1.9 1.3 ± 0.3 12.4 ± 1.2  MI-711

657.02 (4.56) >5  4.8 ± 1.1  9.3 ± 2.5 8.6 ± 1.2 6.3 ± 2.9 MI-712

614.96 (3.82) <3  1.0 ± 0.0  7.9 ± 0.2 1.8 ± 0.4 9.1 ± 1.0 MI-713

706.55 (4.19) <10  2.7 ± 0.7  6.1 ± 2.1 4.9 ± 1.4 4.3 ± 0.7 MI-714

720.56 (4.14) >10  4.3 ± 0.0  7.7 ± 1.6 8.6 ± 0.1 7.5 ± 0.3 MI-715B

690.47 <5  3.0 ± 0.7 >10 2.7 ± 1.0 >10 MI-715C

690.47 <5  2.2 ± 1.2 >10 2.9 ± 0.6 >10 MI-716A

692.52 <3  1.1 ± 0.5 11.5 ± 0.5 1.0 ± 0.5 >10 MI-716B

692.52 <1 0.86 ± 0.3  9.0 ± 2.8 0.6 ± 0.2 11.5 ± 0.7  MI-716C

692.52 <1  0.8 ± 0.1  8.0 ± 2 0.7 ± 0.2 12.0 ± 3.4  MI-717

680.52 >3  2.7 ± 0.6 31.5 ± 1.4 2.2 ± 0.1 47.8 ± 1.4  MI-718

682.05 >3  3.7 ± 0.3 34.2 ± 0.4 3.0 ± 1.1 47.2 ± 5.0 MI-719

730.60 >10    51.2 >100 58.6 ± 14.9 >100 MI-720

680.52 <1 0.76 ± 0.1 15.2 ± 3.2 0.69 ± 0.0  19.5 ± 5.6  MI-721

807.10 <3  1.4 ± 0.0 15.2 ± 1.4 1.5 ± 0.3 18.4 ± 5.4  MI-722

821.13 >100 >100 >100 >100 MI-723

695.22 <5    2.301    24.65 2.422 51.31 MI-724

708.57 <1  1.2 ± 0.4  3.9 ± 0.2 1.1 ± 0.5 5.3 ± 0.3 MI-725

708.57 <1  0.8 ± 0.1  4.7 ± 1.0 0.7 ± 0.0 5.8 ± 2.9 MI-726

722.59 <5  2.3 ± 0.3  5.1 ± 1.4 2.3 ± 0.8 8.0 ± 0.8 MI-727

648.50 >10 12.2 ± 0.5 21.4 ± 4.8 24.1 ± 0.7  20.9 ± 1.1  MI-728

722.59 <5  2.1 ± 0.1  7.1 ± 2.0 4.0 ± 0.8 16.9 ± 5.6  MI-729

694.54 <1  1.0 ± 0.0  6.1 ± 1.6 0.8 ± 0.2 8.9 ± 0.1 MI-730

865.63 <1  1.7 ± 0.5  6.3 ± 1.4 1.4 ± 0.3 7.8 ± 0.7 MI-731

726.56 <1  0.8 ± 0.5  5.6 ± 1.9 0.9 ± 0.5 6.4 ± 1.7 MI-732

710.10 <1  0.7 ± 0.0  5.8 ± 0.0 0.7 ± 0.1 10.2 ± 4.8  MI-733

740.58 >1  2.2 ± 0.5  5.4 ± 1.7 2.4 ± 0.3 8.7 ± 0.9 MI-734

724.13 >5  3.7 ± 1.1  7.4 ± 1.8 5.5 ± 2.1 8.2 ± 3.5 MI-735

692.11 <1  1.1 ± 0.4  8.6 ± 0.9 1.2 ± 0.3 8.5 ± 1.1 MI-736

710.10 <1  0.9 ± 0.0  6.3 ± 1.2 1.0 ± 0.2 4.4 ± 0.9 MI-737

706.14 >1    3.53    3.73    11.3    11.6 5.0 4.5 24.4 15.0 MI-738

724.13 >3  4.8 ± 1.0  8.5 ± 0.6 8.5 ± 2.1 8.2 ± 0.6 MI-743

715.56 >1  2.5 ± 0.5  7.7 ± 3.1 2.4 ± 0.2 5.7 ± 3.4 MI-743B

783.64 >100 >1 >1 >1 >1 MI-753

576.53  3.2 ± 0.3 24.7 ± 2.7 2.9 ± 0.1 21.8 ± 4.9  MI-754

690.55 <1 <3 >5 <3 >5 MI-755

690.55 <1 <3 >10 <3 >10

TABLE 19C Bidning affinities Cell growth inhibition using WST assay toMDM2 as (IC50 value, uM) determined using HCT116 HCT116 Compound FPassay LNCaP PC3 p53WT p53KO ID structure IC₅₀ [uM] IC₅₀ IC₅₀ IC₅₀ IC₅₀MI-319-23

>10 >10 >10 >10 >10 MI-319-24

<1  <5  >10 <5  >10 MI-319-25

<1  <5  >10 <5  >10 MI-319-26

<1  <5  >10 <5  >10 MI-319-27

<5  <5  >10 <5  >10 MI-319-28

<5  <5  >5  <5  >5  MI-319-29

>5  >10 >10 >10 >10 MI-319-30

<5  >5  >10 >10 >10 MI-319-33

<1  1.4 ± 0.2 23.6 ± 5.5  2.1 ± 0.2 25.3 ± 2.8 MI-319-34

<10 <10 >30  9.5 ± 2.1 >30 MI-319-35

<10 3.2 ± 0.3 >10  4.8 ± 0.5 >10 MI-319-36

<5  6.4 ± 1.4 >30 12.2 ± 1.7 >30 MI-319-37

<1  <5  >30 <10 >30 MI-319-38

>3  >5  >10 >10 >10 MI-319-39

>10 >3  >10 >3  >10

TABLE 19D MDM2 HCT116p5 HCT116p5 Binding LNCaP PC3 3WT 3KO AffinitiesIC₅₀ IC₅₀ IC₅₀ IC₅₀ Compounds structure IC₅₀ [uM] (uM) (uM) (uM) (uM)SJSA-1 MI-519-8

<3  <5 >10 <5 >10 MI-519-9

<3  <3 >10 <3 >10 MI-519-10

<3  <3 >10 <3 >10 MI-519-10-me

<10 >1 >30 >1 >30 MI-519-11

>5  >5 >10 >5 >10 MI-519-12

<10 <3 >10 <3 >10 MI-519-13

MI-519-14

<1  <5 >10 <5 >10 MI-519-15

<1  <5 >10 <3 >10 MI-519-15-me

<5  6.4  >10 5.2   >10 MI-219-M8

<1  1.1 ± 0.5 9.2 ± 1.6 1.4 ± 0.9 18.0 ± 4.1 MI-519-20

<5  2.2 ± 0.2 9.0 ± 1.1 1.9 ± 0.1 13.4 ± 0.8 MI-519-21

<3  1.5 ± 0.6 4.8 ± 0.4 1.4 ± 0.5  5.8 ± 1.9 MI-519-22

MI-519-25

<3  <5 >10 <3 >10 MI-519-26

<10 <5 >10 <5 >10 MI-519-52

<1  <1 >3  <1 >5  <1 MI-519-53-1

<1  <1 >3  <1 >3  <1 MI-519-53-2

<1  <1 >3  <1 >3  <1 MI-519-55

<1  <2 >3  <2 >5  <3 MI-519-57-1

<1  <3 8.8 <3 9.5 <3 MI-519-58

<1  <3 >10 <3 >10 <3 MI-519-59

<1  <3 >10 <1 >10 <3 MI-519-66

<3 6.89 <2 5.512 <3 MI-519-67

MI-519-72

<1 

Example 4 Cell Death Assay

Cell death assays were performed using trypan blue staining Cells weretreated in the presence and absence of indicated compounds. Both thefloating and adherent cells were stained with trypan blue. Cells thatstained blue or the morphologically unhealthy cells were scored as deadcells. At least 100 cells were counted in each of three separate areasunder microscope.

As shown in FIGS. 1, 30, and 31 MDM2 inhibitors provided herein inducecell death in SJSA-1 and RS4; 11 cancer cells with wild-type p53.

Example 5 Western Blotting

For Western blot analysis, cells were lyzed in ice-cold RIPA buffer: 20mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% sodiumdeoxycholate, 2.5 mM sodium pyrophosphate, 1 mM b-glycerophosphate, 1 mMsodium orthovanadate and 1 μg/ml leupeptin. The proteins in the wholecell lysates were detected by Western blot analysis using the followingantibodies: anti-p53 (clone DO-1), anti-MDM2 (clone SMP-14), anti-p21(clone SX118), anti-β-actin (clone AC-40) and glyceraldehyde-3-phosphatedehydrogenase (GAPDH; HRP conjugated). As shown in FIGS. 2-4, 27, 28,and 32-25, MDM2 inhibitors provided herein are active in this assay.

Example 6 In Vitro Microsomal Stability

The in vitro stability of the MDM2 inhibitors provided herein wasdetermined using rat and/or human liver microsomes as shown in Tables20-24.

TABLE 20 % of compound remaining when incubated in rat liver microsomesat indicated time (min) compound 0 4 6 10 15 30 MI-519-31 100 80.1 69.454.3 47.7 31 MI-519-38 100 91.5 85.4 82.4 80.8 73.2 MI-519-40 100 84.270.1 59.6 58.7 57.6 MI-519-41 100 84.2 80.4 66.1 55.4 42.9 MI-519-44 10085.6 74.7 66.4 61.2 42.5 MI-519-45 100 81.1 59.6 50.7 44.3 38.5MI-519-46 100 83.8 70.9 53.9 53.4 38.3 MI-519-47 100 80.1 61.1 45.3 38.134.3 MI-518-48 100 79.8 64.8 48.5 45.2 43.7 MI-748 100 79.1 69.7 61.549.9 43.7 MI-749 100 82.9 58.4 54.2 42.7 23 MI-751 100 83.1 62.9 50.141.6 31.1 MI-752 100 84.3 70.3 60.6 52.2 46.4 MI-763 100 96.8 97.3 85.672.1 61.5 MI-764 100 87.8 76.5 66.5 63.9 55.8

TABLE 21 % of compound remaining when incubated in rat liver microsomeswith NADPH at indicated time (min) (min) MI-519- MI-519- MI-519- MI-519-MI-519- MI-519- Time MI-758 19 23 24 27 28 29 (TFA) 0 100.00% 100.00%100.00% 100.00% 100.00% 100.00% 100.00% 5 96.52% 8.14% 43.92% 102.16%57.28% 92.34% 93.42% 10 92.93% 1.61% 34.46% 86.33% 31.78% 87.59% 84.91%15 82.50% 0.64% 18.06% 75.72% 19.34% 86.13% 82.81% 30 73.32% 0.00% 9.01%68.24% 5.81% 71.97% 70.75% 45 69.33% 0.00% 5.70% 66.08% 2.49% 65.40%71.89% 60 66.93% 0.00% 4.76% 61.51% 2.13% 61.79% 69.91% t_(1/2) 79.821.38 6.5 48.52 6.05 74.56 61.30 % of compound remaining when incubatedin rat liver microsomes with NADPH at indicated time (min) (min) MI-519-MI-519- Time 31 MI-771 MI-772 MI-773 51 AT-219  0 100.00% 100.00%100.00% 100.00% 100.00% 100.00% 5 80.22% 20.48% 93.46% 81.32% 12.77%84.05% 10 65.69% 7.59% 91.15% 60.00% 2.84% 60.28% 15 57.57% 3.57% 89.04%56.58% 0.83% 56.91% 30 42.91% 1.22% 88.27% 46.11% 0.00% 52.38% 45 34.31%0.66% 78.65% 41.18% 0.00% 28.04% 60 32.81% 0.46% 75.96% 33.26% 0.00%18.59% t_(1/2) 18.69 2.69 >60 17.28 2.18 27.94

TABLE 22 % of compound remaining when incubated in rat liver (min)microsomes with NADPH at indicated time (min) Time MI-519-63 MI-519-60MI-519-64 MI-519-31 0  100%  100%  100% 100.00% 5 66.01% 79.93% 71.95%80.22% 10 53.79% 63.72% 64.86% 65.69% 15 46.44% 51.52% 48.47% 57.57% 3029.41%  40.6% 43.38% 42.91% 45 27.72% 35.16% 41.19% 34.31% 60 23.28%29.15% 43.03% 32.81%

TABLE 23 % of compound remaining when incubated in human livermicrosomes with NADPH at indicated time (min) (min) MI-519- MI-519-MI-519- MI-519- MI-519- MI-519- Time MI-758 19 23 24 27 28 29 0 100.00%100.00% 100.00% 100.00% 100.00% 100.00% 100.00% 5 95.88% 9.68% 30.98%57.08% 52.96% 91.87% 70.67% 10 89.21% 0.89% 13.14% 53.92% 25.03% 89.02%56.31% 15 81.61% 0.00% 2.60% 49.62% 11.38% 77.40% 49.52% 30 65.20% 0.00%0.55% 47.41% 2.30% 66.83% 45.48% 45 54.52% 0.00% 0.17% 41.18% 1.42%55.85% 40.08% 60 51.45% 0.00% 0.00% 41.42% 0.77% 53.78% 35.28% t_(1/2)49.47 1.48 3.42 11.24 5.00 54.11 12.07 % of compound remaining whenincubated in human liver microsomes with NADPH at indicated time (min)(min) MI-519- MI-519- Time 31 MI-771 MI-772 MI-773 51 AT-219  0 100.00%100.00% 100.00% 100.00% 100.00% 100.00% 5 78.11% 27.98% 95.37% 83.70%36.05% 90.98% 10 58.15% 10.82% 86.87% 82.97% 9.88% 71.93% 15 45.42%4.30% 82.24% 69.38% 3.95% 59.84% 30 22.75% 0.82% 77.91% 52.36% 0.00%43.20% 45 13.25% 0.00% 63.28% 42.57% 0.00% 18.69% 60 8.56% 0.00% 56.87%28.77% 0.00% 10.41% t_(1/2) 13.01 3.12 >60 31.51 3.15 23.96

TABLE 24 % of compound remaining when incubated in human (min) livermicrosomes with NADPH at indicated time (min) Time MI-519-63 MI-519-60MI-519-64 MI-519-31 0 100.00%   100%  100% 100.00% 5 77.75% 83.26%83.66% 78.11% 10 59.66% 68.14% 80.61% 58.15% 15 50.42% 51.54% 65.86%45.42% 30 31.21% 49.28% 61.26% 22.75% 45 26.74% 40.97%  55.9% 13.25% 60 21.5% 37.55% 53.21% 8.56%

Example 7 Pharmacokinetic Studies in Rats

Pharmacokinetic studies on MDM2 inhibitors were carried out in rat. Forcomparison, the MDM2 inhibitor, AT-219, was also studied. As shown inFIGS. 5 and 6, and summarized in Tables 25 and 26, MDM2 inhibitorsprovided herein have promising ADME properties in rat. In particular,MI-773 displays high oral bioavailability; MI-772 displays a long plasmahalf-life. MI-774, the amino acid ester prodrug of MI-773, also has goodoral bioavailability. Of note, in the pharmacokinetic evaluation ofMI-774, the concentration of MI-773 was measured.

TABLE 25 IV dosing in rat. Plasma concentrations of the parent compoundwere measured with the exception of MI-774. MI-774 is an amino esterprodrug of MI-773. Accordingly, the plasma concentrations of MI-773 weremeasured for PK data of MI-774. Dosing (IV, AUC(0 −>t) t½z CLz Vz Ratmg/kg) (μg/L*hr) (hr) (L/h/kg) (L/kg) AT-219 5 2349 2.2 2.13 6.7MI-519-51 5 1110 1.2 4.5 7.5 MI-771 5 2200 2.2 2.2 7.1 MI-772 5 4577 7.41.0 10.9 MI-773 5 4630 2.0 1.0 3.0 MI-774 5 4195 1.6 MI-519-64 5 26693.5 1.8 9.0

TABLE 26 Oral dosing in rat. Plasma concentrations of the parentcompound were measured with the exception of MI-774. MI-774 is an aminoester prodrug of MI-773. Accordingly, the plasma concentrations ofMI-773 were measured for PK data of MI-774. Dosing (oral, AUC (0 -> t)t½z Tmax Cmax F Rat mg/kg) (μg/L * hr) (hr) (hr) (μg/L) (%) AT-219 257677 ± 328 1.4 ± 0.1 0.6 ± 0.4 3752 ± 1068 65 ± 3 MI-519-51 25 1463 ±463 1.1 ± 0.1 1.0 ± 0.0 740 ± 219 26 ± 8 MI-771 25 677 ± 41 1.6 ± 0.10.8 ± 0.3 241 ± 11   6.2 ± 0.4 MI-772 25  3593 ± 2410 8.8 ± 4.2 1.0 ±0.0 445 ± 311 16 ± 9 MI-773 25 17230 ± 4330 1.6 ± 0.1 1.3 ± 0.6 4547 ±809   75 ± 17 MI-774 25 15115 ± 1303 1.5 ± 0.3 2.0 ± 0.0 3177 ± 142  75± 8 MI-519-64 25  7742 ± 1317 3.3 ± 1.4 1.3 ± 0.6 1317 ± 82  56 ± 9

Example 8 In Vivo Efficacy Studies Using SJSA-1 Xenograft Model

SJSA-1 tumor cells were harvested with Trypsin (0.05%)-EDTA (0.53 mM)(GIBCO™, Invitrogen Corp.), growth medium added and cells placed on ice.A cell sample was mixed 1:1 with Trypan Blue (GIBCO™, Invitrogen Corp.)and counted on a hemocytometer to determine the number of live/deadcells. Cells were washed once with 1×PBS (GIBCO™, Invitrogen Corp.) andresuspended in PBS. For Matrigel injections, after washing in PBS, cellsare resuspended in an ice cold mixture of 1:1 PBS and Matrigel (BDBiosciences, Invitrogen Corp.) for a final Matrigel proteinconcentration of 5 mg/ml. SJSA-1 tumors were inoculated into C.B-17 SCIDmice at 5×10⁶ cells in 0.1 ml with Matrigel. Cells were injected s.c.into the flank region of each mouse using a 27 gauge needle.

The size of tumors growing in the mice was measured in two dimensionsusing calipers. Tumor volume (mm³)=(A×B2)/2 where A and B are the tumorlength and width (in mm), respectively. During treatment, tumor volumeand body weight was measured three times a week. After the treatment wasstopped, tumor volume (FIGS. 7, 9, 11, and 36) and body weight (FIGS. 8,10, 12, and 37) was measured at least once a week. Mice were kept for anadditional 60 days for further observation of tumor growth and toxicity.

Before treatment began, tumors were allowed to grow to 60-140 mm³ involume, at which point the blood vessel supplies to the tumor shouldhave been established. Mice with tumors within acceptable size rangewere randomized into treatment groups of 8 mice for experimentalcompounds and 10 mice for the Control group. Experimental compounds weregiven orally, once per day for 2-3 weeks. The Control group receivedvehicle alone (10% PEG 400: 3% Cremophor: 87% PBS). Statistical analysesof the in vivo efficacy of MDM2 inhibitors on SJSA-1 tumors arepresented in Tables 27-29.

These data indicate good in vivo efficacy for certain MDM2 inhibitors ofthe disclosure, particularly MI-519-63, MI-773 and MI-519-64, withoutsignificant weight loss in the animals.

TABLE 27 Exp. No. 1 % Tumor Growth t-test P value t-test P value Inhib.T/C Control vs: Control vs: Control End of Control PO dose 750 mm³ D 27Treatment D 24 750 mm³ D 27 MI-219 44.3% <0.0001 0.0004 300 mg/kgMI-519-49 63.8% <0.0001 0.0028 100 mg/kg MI-519-50 41.1% <0.0001 <0.0001100 mg/kg MI-519-51 34.0% <0.0001 <0.0001 100 mg/kg

TABLE 28 Exp. No. 2 % Tumor Growth t-test P value t-test P value Inhib.T/C Control vs: Control vs: Control End of Control PO dose 750 mm³ D 24Treatment D 31 750 mm³ D 24 MI-219 30.1% <0.0001 <0.0001 300 mg/kgMI-519-62 59.5% 0.2122 0.0015 100 mg/kg MI-519-63 0.0% <0.0001 <0.0001100 mg/kg MI-772 71.1% 0.5673 0.0093 100 mg/kg MI-773 3.2% <0.0001<0.0001 100 mg/kg MI-774 21.4% <0.0001 <0.0001 100 mg/kg MI-779 66.1%0.0487 0.0015 100 mg/kg MI-781 75.5% 0.6687 0.0554 100 mg/kg

TABLE 29 Exp. No. 3 % Tumor Growth t-test P value t-test P value Inhib.T/C Control vs: Control vs: Control End of Control PO dose 750 mm³ D 29Treatment D 31 750 mm³ D 29 MI-219 15.8% <0.0001 <0.0001 300 mg/kgMI-519-64 5.3% <0.0001 <0.0001 100 mg/kg MI-519-64 20.7% <0.0001 <0.000130 mg/kg MI-773 1.2% <0.0001 <0.0001 100 mg/kg MI-773 24.3% <0.0001<0.0001 30 mg/kg

Example 9 Synthesis of MI-519-64 and MI-519-65

Step 1: benzyl 3-oxocyclobutanecarboxylate (2)

BnBr was added to the mixture of compound 1 and K₂CO₃ in acetonitrile150 mL. The mixture was stirred at room temperature over 24 h and thesolid was filtered. The solvent was removed and the residue was purifiedby column chromatography to give compound 2.

Step 2: benzyl 3-hydroxy-3-methylcyclobutanecarboxylates (3 and 4)

MeMgCl in THF was added dropwise to the solution of compound 2 indiethyl ether at −78° C. and the mixture was stirred at the sametemperature for half an hour. After TLC monitoring showed thedisappearance of the starting material, the reaction was quenched byadding aqueous NH₄Cl solution. The aqueous phase was extracted withethyl acetate three times and the combined organic phase was washed withbrine and dried (Na₂SO₄). The solid was filtered and the solvent wasremoved. The residue was purified by column chromatography to givecompounds 3 and 4 (5:1 based on TLC analysis).

Step 3: benzyl 3-(tert-butyldimethylsilyloxy)-3-methylcyclobutanecarboxylates (5 and 6)

To the mixture of compounds 3 and 4 in DMF (10 mL) was added immidazoleand TBSCl, and the resulting mixture was stirred at 80° C. for 30 h.After cooling to room temperature, water was added and the aqueous phasewas extracted with ethyl acetate three times. The combined organic phasewas washed with brine and dried (Na₂SO₄). The solid was filtered and thesolvent was removed. The residue was purified by column chromatographyto get compounds 5 and 6.

Step 4: 3-(tert-butyldimethylsilyloxy)-3-methylcyclobutanecarboxylicacids (7 and 8)

To the mixture of compounds 5 and 6 in isopropanol was added Pd/C. Theresulting mixture was stirred under 1 atm hydrogen for 1 h. TLC showedthe disappearance of the starting material and the solid was filtered.The solvent was removed to give compounds 7 and 8.

Step 5:benzyl-3-(tert-butyldimethylsilyloxy)-3-methylcyclobutylcarbamates 9 and10

To a 0° C. stirring solution of compounds 7 and 8 and Et₃N in acetonewas added ClCOOEt dropwise. The resulting mixture was stirred at 0° C.for 30 min. A solution NaN₃ in water was added, and the resultingmixture was stirred at 0° C. for an additional 20 min. Water was added,and the aqueous phase was extracted with ethyl acetate three times. Thecombined organic phase was washed with brine and dried (Na₂SO₄). Thesolvent was removed and the residue was dissolved in toluene. Benzylalcohol and NaHCO₃ were added. The resulting mixture was stirred at 80°C. for 2 h. All the solvent was removed and the residue was purified bycolumn chromatography to obtain two isomers 9 and 10 in a 5:1 ratio.

Step 6: 3-(tert-butyldimethylsilyloxy)-3-methylcyclobutanamine (11)

To a mixture of the major isomer 9 and NaHCO₃ in isopropanol was addedPd/C and the resulting mixture was stirred under 1 atm hydrogen for 1 h.The solid was filtered and the solvent was removed to give compound 11.

Step 7: 3-(tert-butyldimethylsilyloxy)-3-methylcyclobutanamine (12)

To a mixture of the minor isomer 10 and NaHCO₃ in isopropanol was addedPd/C and the resulting mixture was stirred under 1 atm hydrogen for 1 h.The solid was filtered and the solvent was removed to give compound 12.

Step 8: MI-519-64

To a solution of compound 11 in THF was added compound 13 and theresulting solution was stirred overnight. The solvent was removed andthe residue thus obtained was dissolved in CH₃CN/H₂O (1:1). CAN wasadded and the reaction mixture was stirred for 30 min. Water was addedand the aqueous phase was extracted with ethyl acetate three times. Thecombined organic layers were dried (Na₂SO₄), filtered, and concentrated.The residue was purified by column chromatography on silica gel to givecompound 14. Compound 14 was dissolved in methanol, 12M HCl in water wasadded, and the reaction mixture was stirred for 1 h at room temperature.The solvent was removed and the residue was purified by HPLC to giveMI-519-64 as the TFA salt. ¹H NMR (300 MHz, CD₃OD) δ 7.54-7.52 (m, 1H),7.42-7.38 (m, 1H), 7.23-7.18 (m, 1H), 6.88-6.75 (m, 3H), 5.04 (d, J=9.9Hz, 1H), 4.45 (d, J=9.9 Hz, 1H), 4.19-4.16 (m, 1H), 3.92-3.89 (m, 1H),2.42-2.11 (m, 2H), 2.10-1.87 (m, 3H), 1.32-1.24 (m, 4H), 0.82 (s, 9H);MS (ESI) m/z 548 [M+H]⁺.

Step 9: MI-519-65

To a solution of compound 12 in THF was added compound 13 and theresulting solution was stirred overnight. The solvent was removed andthe residue was dissolved in CH₃CN/H₂O (1:1). CAN was added and thereaction mixture was stirred for 30 min. Water was added and the aqueousphase was extracted with ethyl acetate three times. The combined organiclayers were dried (Na₂SO₄), filtered, and concentrated. The residue waspurified by column chromatography on silica gel to give compound 15.Compound 15 was dissolved in methanol, 12M HCl in water was added, andthe reaction mixture was stirred for 1 h at room temperature. Thesolvent was removed and the residue was purified by HPLC to giveMI-519-65 as the TFA salt. ¹H NMR (300 MHz, CD₃OD) δ 7.50 (m, 1H),7.44-7.38 (m, 1H), 7.24-7.20 (m, 1H), 6.89-6.88 (m, 1H), 6.80 (m, 1H),6.71 (m, 1H), 4.91-4.88 (m, 1H), 4.40-4.36 (m, 2H), 4.10-4.06 (m, 1H),2.41-2.33 (m, 2H), 2.07-1.87 (m, 3H), 1.25-1.21 (m, 4H), 0.82 (s, 9H);MS (ESI) m/z 548 [M+H]⁺.

Example 11 Synthesis of MI-519-6401

MI-519-64 (100 mg) purified by flash chromatography on silica gel wasplaced in a 50 mL round-bottom-flask equipped with magnetic stirringbar. Acetonitrile (20 mL) was added to fully dissolve the compound anddeionized water (7 to 10 mL) was added. NaHCO₃ saturated aqueoussolution (ca 0.5 mL) was then added to adjust the pH value between 7 and8. This solution was allowed to stir at room temperature for at least 12h. TFA (0.1 mL) and another 10 mL of deionized water were added to thesolution and the solution was purified by semi-preparative RP-HPLCimmediately using acetonitrile and water as the eluents to giveMI-519-6401 as the TFA salt. ¹H NMR (300 MHz, MeOH-d4): 7.62-7.53 (m,2H), 7.45-7.35 (m, 1H), 7.20-7.10 (m, 2H), 6.80-6.85 (m, 1H), 5.11 (d,J=11.07 Hz, 1H), 4.57 (d, J=11.11 Hz, 1H), 4.40 (d, J=7.39 Hz, 1H),4.00-3.80 (m, 1H), 2.50-2.35 (m, 1H), 2.35-2.20 (m, 1H), 2.10-1.90 (m,1H), 1.90-1.60 (m, 2H), 1.30 (s, 3H), 1.20-1.05 (m, 1H), 0.88 (s, 9H);¹³C NMR (75 MHz, MeOH-d4): 177.8, 168.0, 157.6 (d, J_(C-F)=249 Hz),144.9, 136.8, 132.2, 128.5, 126.5, 126.3 (d, J_(C-F)=4.76), 123.9,123.7, 122.3 (d, J_(C-F)=18.97 Hz), 122.0 (d, J_(C-F)=13.1 Hz), 111.8,67.1, 64.6, 64.5, 62.9, 49.0, 45.5, 45.4, 43.3, 38.0, 30.8, 29.5, 27.3;ESI-MS calculated for C₂₈H₃₃ ³⁵Cl₂FN₃O₃ [M+H]⁺: 548.1883. Found: 548.25.

Analytical RP-HPLC spectra are presented in FIGS. 20-22. Referring toFIG. 21, MI-519-6401 corresponds to the RP-HPLC peak at 31.787 minutes.

In an alternate procedure, MI-519-64 (100 mg) purified by flashchromatography on silica gel was placed in a 50 mL round-bottom-flaskequipped with magnetic stirring bar. Methanol (20 mL) was added to fullydissolve the compound and deionized water (10 to 20 mL) was added.NaHCO₃ saturated aqueous solution (ca 0.5 mL) was then added to adjustthe pH value between 7 and 8. This solution was allowed to stir at roomtemperature for at least 12 h. TFA (0.1 mL) and another 10 mL ofdeionized water were added to the solution and the solution was purifiedby semi-preparative RP-HPLC immediately using acetonitrile and water asthe eluents to give MI-519-6401 as the TFA salt.

C02701, C02901, C03001, C03401, C03701, and C04801 of Example 1 wereprepared using procedures similar to that used to prepare MI-519-6401.

Example 11 Synthesis of MI-758

Step 1: ((3-methylbut-3-enyloxy)methyl)benzene (1)

BnBr was added to the mixture of alcohol and NaH in DMF cooled in an icebath. The mixture was stirred at room temperature overnight and thesolid was filtered. The solvent was removed and the residue was purifiedby column chromatography to give compound 1.

Step 2: (S)-4-(benzyloxy)-2-methylbutane-1,2-diol (2)

Compound 1 was added to a solution of AD-mix-α in tBuOH/H₂O (1:1) cooledin an ice bath. After TCL monitoring showed the disappearance of thestarting material, the reaction was quenched by adding Na₂SO₃. Theaqueous phase was extracted with ethyl acetate three times and thecombined organic phase was washed with brine and dried (Na₂SO₄). Thesolid was filtered and the solvent was removed. The residue was purifiedby column chromatography to give compound 2.

Step 3: (S)-4-(2-(benzyloxy)ethyl)-2,2,4-trimethyl-1,3-dioxolane (3)

To the mixture of compound 2 and 2,2-dimethoxypropane in DCM was addedp-TSA. After TLC monitoring showed the disappearance of the startingmaterial, aqueous NaHCO₃ solution was added and then extracted withethyl acetate three times. The combined organic phase was washed withbrine and dried (Na₂SO₄). The solid was filtered and the solvent wasremoved. The residue was purified by column chromatography to givecompound 3.

Step 4: (S)-2-(2,2,4-trimethyl-1,3-dioxolan-4-yl)ethanol (4)

To a solution of 3 in MeOH was added Pd/C. The resulting mixture wasstirred under 1 atm hydrogen overnight. TLC showed the disappearance ofthe starting material and the solid was filtered. The solvent wasremoved to give compound 4.

Step 5: (S)-2-(2,2,4-trimethyl-1,3-dioxolan-4-yl)ethyl 4-methylbenzenesulfonate (5)

To a solution of compound 4 and Et₃N in DCM was added TsCl dropwise. Theresulting mixture was stirred at 0° C. for 30 min. The solid wasfiltered and the solvent was removed. The residue was purified by columnchromatography to give compound 5.

Step 6: (S)—N-benzyl-2-(2,2,4-trimethyl-1,3-dioxolan-4-yl)ethanamine (6)

A solution of 5 and BnNH₂ in THF was heated to reflux overnight. Thesolvent was removed. The residue was purified by column chromatographyto give compound 6.

Step 7: (S)-2-(2,2,4-trimethyl-1,3-dioxolan-4-yl)ethanamine (7)

To a solution of 6 in MeOH was added Pd/C. The resulting mixture wasstirred under 1 atm hydrogen overnight. TLC showed the disappearance ofthe starting material and the solid was filtered. The solvent wasremoved to give compound 7.

Step 8: MI-758 (11)

To a solution of compound 7 in THF was added compound 8 and theresulting solution was stirred overnight. The solvent was removed andthe residue was dissolved in CH₃CN/H₂O (1:1). CAN was added and thereaction mixture was stirred for 5 min. K₂CO₃ was added and the aqueousphase was extracted with ethyl acetate three times. The combined organiclayers were dried (Na₂SO₄), filtered, and concentrated. The residue waspurified by column chromatography on silica gel to give compound 11.Compound 11 was dissolved in dioxane, 4M HCl in dioxane was added, andthe reaction mixture was stirred for 0.5 h at room temperature. Thesolvent was removed and the residue was purified by HPLC to give MI-758as the TFA salt. ¹H NMR (300 MHz, CD₃OD) δ 8.44 (s, 1H), 7.72 (d, J=8.4Hz, 1H), 7.19-7.36 (m, 3H), 7.09 (d, J=7.5 Hz, 1H), 6.89 (d, J=6.0 Hz,1H), 5.28 (d, J=11.1 Hz, 1H), 4.50 (d, J=6.6 Hz, 1H), 4.16 (d, J=11.1Hz, 1H), 3.30 (s, 2H), 1.94 (dd, J=8.4, 15.3 Hz, 1H), 1.47-1.68 (m, 2H),1.18 (d, J=15.6 Hz, 1H), 1.07 (s, 3H), 0.93 (s, 9H).

Example 12 Synthesis of MI-773

Step 1

To a stirred solution of oxindole 1 (4.19 g, 25 mmol) in methanol (50mL) was added aldehyde 2 (3.96 g, 25 mmol) and piperidine (2.45 mL, 25mmol). The reaction mixture was stirred at room temperature for 3 h andthe yellow precipitate was collected, washed successively with methanol,hexanes, and ethyl ether and dried to give compound 3 (6.25 g, 81%yield).

Step 2

To a solution of compound 3 (6.25 g, 21 mmol) in toluene (75 ml) wasadded compound 4 (5.43 g, 21 mmol), compound 5 (2.15 g, 21 mmol) and 4 Åmolecular sieves (4 g). The reaction mixture was heated at refluxovernight and filtrated. The filtrate was evaporated and the residue waspurified by silica gel flash column chromatography (n-hexane/ethylacetate=9:1 to 5:1) to give compound 6 (8.78 g, 65% yield).

Step 3

The solution of compound 6 (965 mg, 1.5 mmol) and amine 7 (346 mg, 3mmol) in 5 mL of THF was stirred at room temperature for 2 days and thesolvent was removed under reduced pressure. The residue was purified bysilica gel flash column chromatography (n-hexane/ethyl acetate=1:1 to1:4) to give compound 8 (819 mg, 72% yield).

Step 4

To an ice-bath cooled solution of compound 8 (800 mg, 1.05 mmol) inCH₃CN (8 ml), H₂O (4 ml) and acetone (4 ml) was added CAN (ammoniumcerium) (1.15 g, 2.1 mmol). Progress of the reaction was monitored byTLC. When all the starting material disappeared (around 5 min), 100 mgof NaHCO₃ powder was added and the reaction mixture was diluted with 50mL of ethyl acetate. The organic phase was dried over anhydrous Na₂SO₄,filtered, and concentrated. The residue was purified by silica gel flashcolumn chromatography (methylene chloride/methanol/triethylamine=200:1:1to 200:10:1) to give(2′R,3S,4′S,5′R)-6-chloro-4′-(3-chloro-2-fluorophenyl)-N-((trans-4-hydroxycyclohexyl)-2′-neopentyl-2-oxospiro[indoline-3,3′-pyrrolidine]-5′-carboxamide(MI-773) (402 mg, 68% yield). The absolute stereochemical configurationof MI-773 was determined by x-ray analysis.

MI-773 was dissolved in DCM and added TFA and then evaporated. Theresidue was further purified by chromatography on a C18 reverse phasesemi-preparative HPLC column with solvent A (0.1% of TFA in water) andsolvent B (0.1% of TFA in methanol) as eluents (gradient: 45% of solventA and 55% of solvent B to 30% of solvent A and 70% of solvent B in 30min) to give MI-773 as the TFA salt. NMR for MI-773 TFA: ¹H NMR (300MHz, CD₃OD) δ 7.47 (t, J=7.0 Hz, 1H), 7.34 (t, J=7.4 Hz, 1H), 7.14 (t,J=7.9 Hz, 1H), 6.83 (s, 1H), 6.80 (s, 2H), 4.39 (d, J=10.0 Hz, 1H),4.15-4.05 (m, 1H), 3.72-3.53 (m, 1H), 3.53-3.85 (m, 2H), 2.10-1.75 (m,4H), 1.62 (d, J=12.2 Hz, 1H), 1.45-1.05 (m, 5H), 0.78 (s, 9H).

Stability of MI-773 (TFA salt): MI-773 (TFA salt) was dissolved in awater/methanol mixture (water/methanol=1:1 with 0.1% of TFA). Thesolution was allowed to stand at room temperature. The purity was testedusing a C18 reverse phase analytical HPLC column at the time points of0, 12 h, 24 h, 48 h, and 72 h. The results showed transformation ofMI-773 to three compounds having the same molecular weight (FIG. 25).The purity of an identical sample solution stored at 4° C. was alsotested at 0 and 36 h. The results showed comparably slow transformationof MI-773 at 4° C.

Example 14 Synthesis of MI-77301

MI-773 (as the TFA salt) was dissolved in MeOH/H₂O (1:1 v/v ratio) andallowed to stand at room temperature for 1-4 days. The solution waspurified by chromatography on a C18 reverse phase semi-preparative HPLCcolumn with solvent A (0.1% of TFA in water) and solvent B (0.1% of TFAin methanol) as eluents (gradient: 45% of solvent A and 55% of solvent Bto 30% of solvent A and 70% of solvent B in 30 min). MI-77301 wasisolated as the TFA salt. ¹H NMR (300 MHz, MeOH-d4): 8.35 (d, J=7.8 Hz,1H), 7.54-7.62 (m, 2H), 7.37-7.43 (m, 1H), 7.12-7.20 (m, 2H), 6.80 (d,J=1.5 Hz, 1H), 5.20 (d, J=11.4 Hz, 1H), 4.58 (d, J=11.4 Hz, 1H), 4.51(d, J=7.2 Hz, 1H), 3.50-3.75 (m, 1H), 3.30-3.50 (m, 1H), 1.82-2.00 (m,3H), 1.76 (d, J=10.5 Hz, 1H), 1.52 (d, J=12.3 Hz, 1H), 1.05-1.42 (m,4H), 0.88-1.00 (m, 1H), 0.88 (s, 9H); ¹³C NMR (75 MHz, MeOH-d4): 177.7,166.9, 157.6 (d, JC-F=248.0 Hz), 145.0, 137.0, 132.4, 128.6, 126.6,126.4 (d, JC-F=4.9), 124.0, 123.4, 122.3 (d, JC-F=18.8 Hz), 121.5 (d,JC-F=12.8 Hz), 111.9, 69.9, 64.4, 64.0, 62.8, 49.7, 34.3, 34.2, 30.9,30.82, 30.77, 29.4; Purity (HPLC): >95%.

In an alternative procedure, MI-773 (77 mg) was dissolved in 15 mLMeOH/H₂O (v/v=1:1). After 3 days, the needle crystals that had formedwere collected, washed with cold MeOH/H₂O (v/v=1:1) and dried in vacuumto give MI-77301 as the free amine (20 mg; >95% purity as determined byHPLC). ¹H NMR (300 MHz, MeOH-d4): 7.49-7.55 (m, 1H), 7.25-7.31 (m, 1H),7.10-7.16 (m, 1H), 6.82 (d, J=1.8 Hz, 1H), 6.50-6.71 (m, 1H), 6.49 (d,J=8.4 Hz, 1H), 4.32 (d, J=9.0 Hz, 1H), 4.09 (d, J=8.7 Hz, 1H), 3.57-3.69(m, 1H), 3.49 (d, J=9.2 Hz, 1H), 3.46-3.57 (m, 1H), 1.83-2.07 (m, 3H),1.68-1.80 (m, 1H), 1.54 (dd, J=9.0, 14.3 Hz, 1H), 1.12-1.45 (m, 5H),0.80 (s, 9H). The absolute stereochemical configuration of MI-77301 wasdetermined by X-ray analysis.

Stability of MI-77301 (TFA salt): MI-77301 (TFA salt) was dissolved in awater/methanol mixture (water/methanol=1:1 with 0.1% of TFA). Thesolution was allowed to stand at room temperature. The purity was testedusing a C18 reverse phase analytical HPLC column at the time points of0, 12 h, 48 h, and 72 h. The results showed slow transformation ofMI-77301 to three compounds having the same molecular weight (FIG. 26).

MI-71201, MI-710401, MI-710501, and MI-710901 of Example 1 were preparedusing procedures similar to that used to prepare MI-519-6401.

Having now fully described the methods, compounds, and compositions ofmatter provided herein, it will be understood by those of skill in theart that the same can be performed within a wide and equivalent range ofconditions, formulations, and other parameters without affecting thescope of the methods, compounds, and compositions provided herein or anyembodiment thereof. All patents, patent applications and publicationscited herein are fully incorporated by reference herein in theirentirety.

1. A compound having Formula I:

wherein: R^(1a), R^(1b), R^(1c), and R^(1d) are independently selectedfrom the group consisting of hydrogen, halogen, hydroxy, amino, nitro,cyano, alkoxy, aryloxy, optionally substituted alkyl, haloalkyl,optionally substituted cycloalkyl, optionally substituted alkenyl,optionally substituted cycloalkenyl, optionally substituted aryl,optionally substituted heteroaryl, carboxamido, and sulfonamido; R² isselected from the group consisting of optionally substituted aryl andoptionally substituted heteroaryl; R³ is selected from the groupconsisting of optionally substituted alkyl, optionally substituted(cycloalkyl)alkyl, optionally substituted cycloalkyl, optionallysubstituted alkenyl, optionally substituted cycloalkenyl, optionallysubstituted aryl, and optionally substituted heteroaryl; R⁴ is selectedfrom the group consisting of hydrogen and optionally substituted C₁-C₆alkyl; R⁵ is selected from the group consisting of:

wherein: each R^(6a) and R^(6b) is independently selected from the groupconsisting of hydrogen and optionally substituted C₁-C₆ alkyl; R⁷ isselected from the group consisting of hydrogen, optionally substitutedC₁-C₆ alkyl, and optionally substituted cycloalkyl; R^(8a) and R^(8b)are each independently selected from the group consisting of hydrogen,optionally substituted C₁-C₆ alkyl, and optionally substitutedcycloalkyl; or R^(8a) and R^(8b) taken together with the carbon thatthey are attached form a 3- to 8-membered optionally substitutedcycloalkyl; W¹ is selected from the group consisting of −OR^(9a) and—NR^(9b)R^(9c); R^(9a) is hydrogen; R^(9b) is selected from the groupconsisting of hydrogen, optionally substituted alkyl, optionallysubstituted cycloalkyl, optionally substituted aryl, optionallysubstituted heteroaryl, —SO₂R^(9d), and —CONR^(9e)R^(9f); R^(9c) isselected from the group consisting of hydrogen, optionally substitutedalkyl, optionally substituted cycloalkyl, optionally substituted aryl,and optionally substituted heteroaryl; or R^(9b) and R^(9e) takentogether with the nitrogen atom to which they are attached form a 4- to8-membered optionally substituted heterocyclo; R^(9d) is selected fromthe group consisting of optionally substituted alkyl and optionallysubstituted cycloalkyl; R^(9e) and R^(9f) are each independentlyselected from the group consisting of hydrogen, optionally substitutedalkyl, and optionally substituted cycloalkyl; or R^(9e) and R^(9f) takentogether with the nitrogen atom to which they are attached form a 4- to8-membered optionally substituted heterocyclo; W² is selected from thegroup consisting of −OR¹⁰ and —NR^(11a)R^(11b); R¹⁰ is hydrogen; or oneof R^(9a) and R¹⁰ is hydrogen and the other is a metabolically cleavablegroup; with the proviso that when W¹ is −OR^(9a) and W² is −OR¹⁰ then atleast one of R⁷, R^(8a), and R^(8b) is other than hydrogen; R^(11a) isselected from the group consisting of hydrogen, optionally substitutedalkyl, optionally substituted cycloalkyl, optionally substituted aryl,optionally substituted heteroaryl, —SO₂R^(11c), and —CONR^(11d)R^(11e);R^(11b) is selected from the group consisting of hydrogen, optionallysubstituted alkyl, optionally substituted cycloalkyl, optionallysubstituted aryl, and optionally substituted heteroaryl; or R^(11a) andR^(11b) taken together with the nitrogen atom to which they are attachedform a 4- to 8-membered optionally substituted heterocyclo; R^(11c) isselected from the group consisting of optionally substituted alkyl andoptionally substituted cycloalkyl; R^(11d) and R^(11e) are eachindependently selected from the group consisting of hydrogen, optionallysubstituted alkyl, and optionally substituted cycloalkyl; or R^(11d) andR^(11e) together with the nitrogen atom to which they are attached forma 4- to 8-membered optionally substituted heterocyclo; n is 1, 2, 3, 4,or 5; each R^(12a), R^(12b), R^(12c) and R^(12d) is independentlyselected from the group consisting of hydrogen and optionallysubstituted C₁-C₆ alkyl; R¹³ is selected from the group consisting ofhydrogen and optionally substituted C₁-C₆ alkyl; R¹⁴ is selected fromthe group consisting of hydrogen, optionally substituted C₁-C₆ alkyl,and optionally substituted cycloalkyl; Z is selected from the groupconsisting of −OR¹⁵ and —NR^(16a)R^(16b); or Z and R¹⁴ taken togetherform a carbonyl group; R¹⁵ is selected from the group consisting ofhydrogen and metabolically cleavable group; R^(16a) is selected from thegroup consisting of —SO₂R^(16c) and —CONR^(16d)R^(16e); R^(16b) isselected from the group consisting of hydrogen and optionallysubstituted alkyl; R^(16c) is selected from the group consisting ofoptionally substituted alkyl, optionally substituted cycloalkyl,optionally substituted aryl, and optionally substituted heteroaryl;R^(16d) and R^(16e) are each independently selected from the groupconsisting of hydrogen, optionally substituted alkyl, optionallysubstituted cycloalkyl, optionally substituted aryl, and optionallysubstituted heteroaryl; or R^(16d) and R^(16e) taken together with thenitrogen atom to which they are attached form a 4- to 8-memberedheterocyclo; o is 1, 2, or 3; p is 0, 1, 2, or 3; each R^(17a), R^(17b),R^(17c) and R^(17d) is independently selected from the group consistingof hydrogen and optionally substituted C₁-C₆ alkyl; R¹⁸ is selected fromthe group consisting of hydrogen and optionally substituted C₁-C₆ alkyl;R¹⁹ is selected from the group consisting of hydrogen, optionallysubstituted C₁-C₆ alkyl, and optionally substituted cycloalkyl; R²⁰ isselected from the group consisting of hydrogen, optionally substitutedC₁-C₆ alkyl, and optionally substituted cycloalkyl; R^(21a) and R^(21b)are each hydrogen; or one of R^(21a) and R^(21b) is hydrogen and theother is metabolically cleavable group; q is 0, 1, 2, or 3; r is 1, 2,or 3; X is selected from the group consisting of O, S, and NR′; Y isselected from the group consisting of O, S, and NR″; R′ is selected fromthe group consisting of hydrogen, optionally substituted alkyl, aralkyl,and optionally substituted cycloalkyl; R″ is selected from the groupconsisting of hydrogen, optionally substituted alkyl, aralkyl, andoptionally substituted cycloalkyl; and

represents a single or a double bond, or a pharmaceutically acceptablesalt, solvate, or prodrug thereof. 2-40. (canceled)